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Bone marrow endothelium plays an important role in the homing of hematopoietic stem and progenitor cells upon transplantation, but surprisingly little is known on how the bone marrow endothelial cells regulate local permeability and hematopoietic stem and progenitor cells transmigration. We show that temporal loss of vascular endothelial-cadherin function promotes vascular permeability in BM, even upon low-dose irradiation. Loss of vascular endothelial-cadherin function also enhances homing of transplanted hematopoietic stem and progenitor cells to the bone marrow of irradiated mice although engraftment is not increased. Intriguingly, stabilizing junctional vascular endothelial-cadherin in vivo reduced bone marrow permeability, but did not prevent hematopoietic stem and progenitor cells migration into the bone marrow, suggesting that hematopoietic stem and progenitor cells use the transcellular migration route to enter the bone marrow. Indeed, using an in vitro migration assay, we show that human hematopoietic stem and progenitor cells predominantly cross bone marrow endothelium in a transcellular manner in homeostasis by inducing podosome-like structures. Taken together, vascular endothelial-cadherin is crucial for BM vascular homeostasis but dispensable for the homing of hematopoietic stem and progenitor cells. These findings are important in the development of potential therapeutic targets to improve hematopoietic stem and progenitor cell homing strategies.

Objective: Vascular endothelial (VE)-cadherin is of dominant importance for the formation and stability of endothelial junctions, yet induced gene inactivation enhances vascular permeability in the lung but does not cause junction rupture. This study aims at identifying the junctional adhesion molecule, which is responsible for preventing endothelial junction rupture in the pulmonary vasculature in the absence of VE-cadherin. Approach and Results: We have compared the relevance of ESAM (endothelial cell-selective adhesion molecule), JAM (junctional adhesion molecule)-A, PECAM (platelet endothelial cell adhesion molecule)-1, and VE-cadherin for vascular barrier integrity in various mouse tissues. Gene inactivation of ESAM enhanced vascular permeability in the lung but not in the heart, skin, and brain. In contrast, deletion of JAM-A or PECAM-1 did not affect barrier integrity in any of these organs. Blocking VE-cadherin with antibodies caused lethality in ESAM −/− mice within 30 minutes but had no such effect in JAM-A −/− , PECAM-1 −/− or wild-type mice. Likewise, induced gene inactivation of VE-cadherin caused rapid lethality only in the absence of ESAM. Ultrastructural analysis revealed that only combined interference with VE-cadherin and ESAM disrupted endothelial junctions and caused massive blood coagulation in the lung. Mechanistically, we could exclude a role of platelet ESAM in coagulation, changes in the expression of other junctional proteins or a contribution of cytoplasmic signaling domains of ESAM. Conclusions: Despite well-documented roles of JAM-A and PECAM-1 for the regulation of endothelial junctions, only for ESAM, we detected an essential role for endothelial barrier integrity in a tissue-specific way. In addition, we found that it is ESAM which prevents endothelial junction rupture in the lung when VE-cadherin is absent.

Summary Measurements of flow velocities at the level of individual arterial vessels and sinusoidal capillaries are crucial for understanding the dynamics of hematopoietic stem and progenitor cell homing in the bone marrow vasculature. We have developed two complementary intravital two-photon imaging approaches to determine blood flow dynamics and velocities in multiple vessel segments by capturing the motion of red blood cells. High-resolution spatiotemporal measurements through a cranial window to determine short-time dynamics of flowing blood cells and repetitive centerline scans were used to obtain a detailed flow-profile map with hemodynamic parameters. In addition, we observed the homing of individual hematopoietic stem and progenitor cells and obtained detailed information on their homing behavior. With our imaging setup, we determined flow patterns at cellular resolution, blood flow velocities and wall shear stress in small arterial vessels and highly branched sinusoidal capillaries, and the cellular dynamics of hematopoietic stem and progenitor cell homing., Graphical Abstract, Highlights • Detailed 3D reconstruction of the calvarial bone marrow microvasculature • Blood flow dynamics at cellular resolution in bone microvessels • Detailed flow map with hemodynamic parameters in arteries and highly branched sinusoids • Cellular dynamics of HSPC homing to bone marrow sinusoids, Bixel et al. use intravital two-photon imaging to determine blood flow patterns at cellular resolution and hemodynamic parameters in individual arterial vessels and sinusoidal capillaries in the bone marrow microvasculature. They report detailed information on the dynamics of hematopoietic stem and progenitor cell homing to highly branched bone marrow sinusoids.

Endothelial cells line blood and lymphatic vessels and form intercellular junctions, which preserve vessel structure and integrity. The vascular endothelial cadherin, VE‐cadherin, mediates endothelial adhesion and is indispensible for blood vessel development and permeability regulation. However, its requirement for lymphatic vessels has not been addressed. During development, VE‐cadherin deletion in lymphatic endothelial cells resulted in abortive lymphangiogenesis, edema, and prenatal death. Unexpectedly, inducible postnatal or adult deletion elicited vessel bed‐specific responses. Mature dermal lymph vessels resisted VE‐cadherin loss and maintained button junctions, which was associated with an upregulation of junctional molecules. Very different, mesenteric lymphatic collectors deteriorated and formed a strongly hyperplastic layer of lymphatic endothelial cells on the mesothelium. This massive hyperproliferation may have been favored by high mesenteric VEGF‐C expression and was associated with VEGFR‐3 phosphorylation and upregulation of the transcriptional activator TAZ. Finally, intestinal lacteals fragmented into cysts or became highly distended possibly as a consequence of the mesenteric defects. Taken together, we demonstrate here the importance of VE‐cadherin for lymphatic vessel development and maintenance, which is however remarkably vessel bed‐specific.

Growth differentiation factor 15 (GDF-15) is the first cytokine known to counteract chemokine-induced activation of leukocyte integrins. We showed recently that this activity dampens neutrophil recruitment into inflamed tissue and is required for survival of myocardial infarction in mice. The receptor responsible for this GDF-15-triggered anti-inflammatory mechanism on myeloid cells is not known. Here, we identify this receptor as transforming growth factor β receptor I (TGF-βRI) (activin receptor-like kinase 5 [ALK-5]) and TGF-β receptor II (TGF-βRII). We show that interference with these receptors by small-molecule inhibitors, antibodies, or small interfering RNA, blocked the GDF-15 effect on leukocyte integrin activation. Likewise, gene inactivation of each of the 2 receptors in neutrophils isolated from conditional gene-deficient mice abolished the inhibitory effect of GDF-15 on CXCL1-induced β2-integrin activation and neutrophil diapedesis. Rapid neutrophil arrest induced by CXCL1 in vivo was inhibited by GDF-15 in an ALK-5 and TGF-βRII dependent way. As for GDF-15 gene-deficient mice, we found that extravasation of neutrophils deficient for ALK-5 or TGF-βRII was strongly increased in the interleukin-1β inflamed cremaster. The inhibitory effects of GDF-15 on neutrophil integrin activation and in vivo neutrophil arrest were also found for TGF-β1. Mechanistically, GDF-15 and TGF-β1 interfered with integrin activation by inhibiting the activation of Ras-related protein 1 (Rap-1), an effect that depended on CalDAG- guanine nucleotide exchange factor 1 (GEF1) and cell division control protein 42 homolog. We conclude that both GDF-15 and TGF-β1 counteract chemokine-induced integrin activation on neutrophils via the ALK-5/TGF-βRII heterodimer. This represents a novel, rapid anti-inflammatory activity of the 2 TGF-β receptors and of TGF-β1.

Bone marrow (BM) endothelium plays an important role in homing of hematopoietic stem and progenitor cells (HSPCs) upon transplantation, but surprisingly little is known on how endothelial cells regulate local permeability and HSPC transmigration. We show that temporal loss of VE-cadherin function promotes vascular permeability in BM, even upon low dose irradiation and strongly enhanced homing of transplanted HSPCs to BM of irradiated mice. Intriguingly, stabilizing junctional VE-cadherinin vivoreduced BM permeability, but did not prevent HSPC migration into the BM, suggesting that HSPCs enter the bone marrow by transcellularly crossing the endothelium. Indeed, HSPCs induce podosomes to cross human BM endothelial monolayers in a transcellular manner. By contrast, HSPC rather use the paracellular route when VE-cadherin function is inhibited. Taken together, VE-cadherin is crucial for BM vascular homeostasis and HSPC homing, and may therefore serve as a potential therapeutic target to improve HSPC homing strategies.

BACKGROUND. Lack of investigatory and diagnostic tools has been a major contributing factor to the failure to mechanistically understand lymphedema and other lymphatic disorders in order to develop effective drug and surgical therapies. One difficulty has been understanding the true changes in lymph vessel pathology from standard 2D tissue sections. METHODS. VIPAR (volume information-based histopathological analysis by 3D reconstruction and data extraction), a light-sheet microscopy–based approach for the analysis of tissue biopsies, is based on digital reconstruction and visualization of microscopic image stacks. VIPAR allows semiautomated segmentation of the vasculature and subsequent nonbiased extraction of characteristic vessel shape and connectivity parameters. We applied VIPAR to analyze biopsies from healthy lymphedematous and lymphangiomatous skin. RESULTS. Digital 3D reconstruction provided a directly visually interpretable, comprehensive representation of the lymphatic and blood vessels in the analyzed tissue volumes. The most conspicuous features were disrupted lymphatic vessels in lymphedematous skin and a hyperplasia (4.36-fold lymphatic vessel volume increase) in the lymphangiomatous skin. Both abnormalities were detected by the connectivity analysis based on extracted vessel shape and structure data. The quantitative evaluation of extracted data revealed a significant reduction of lymphatic segment length (51.3% and 54.2%) and straightness (89.2% and 83.7%) for lymphedematous and lymphangiomatous skin, respectively. Blood vessel length was significantly increased in the lymphangiomatous sample (239.3%). CONCLUSION. VIPAR is a volume-based tissue reconstruction data extraction and analysis approach that successfully distinguished healthy from lymphedematous and lymphangiomatous skin. Its application is not limited to the vascular systems or skin. FUNDING. Max Planck Society, DFG (SFB 656), and Cells-in-Motion Cluster of Excellence EXC 1003.

CD99-like 2 (CD99L2) is a membrane protein with moderate sequence homology to CD99, which initiates cell aggregation of transfected cells and that is strongly expressed on endothelial cells, neutrophils, and lymphocytes. We showed recently that Abs against CD99L2 inhibit neutrophil, but not T lymphocyte, recruitment into inflamed tissues. In this study, we have generated conditional gene–deficient mice for CD99L2 and show by analyzing them in various inflammation models several results. First, gene ablation of CD99L2 impairs neutrophil recruitment into inflamed cremaster and peritoneum. Second, despite the strong expression of CD99L2 on peripheral neutrophils, only gene ablation on endothelial cells but not on myeloid cells affects neutrophil extravasation. Third, in contrast to our previous Ab-based results, recruitment of activated T cells into inflamed skin was impaired in mice lacking CD99L2 on endothelial cells. We conclude that CD99L2 is an essential endothelial Ag for leukocyte extravasation, which does not require homophilic interactions with CD99L2 on leukocytes.

Background/Aims This study was designed to investigate the role of endothelial cell-selective adhesion molecule (ESAM), a recently discovered receptor expressed in endothelial tight junctions and platelets, for leukocyte migration in inflamed liver. Methods The role of ESAM for leukocyte migration in the liver was analyzed using ESAM-deficient mice in a model of warm hepatic ischemia-reperfusion (90min/30–360min). Results As shown by immunostaining, ESAM is expressed in sinusoids as well as in venules and is not upregulated upon I/R. Emigrated leukocytes were quantified in tissue sections. Postischemic neutrophil transmigration was significantly attenuated in ESAM−/− mice after 2h of reperfusion, whereas it was completely restored after 6h. In contrast, T-cell migration did not differ between ESAM+/+ and ESAM−/− mice. Using intravital microscopy, we demonstrate that ESAM deficiency attenuates I/R-induced vascular leakage after 30min of reperfusion. The I/R-induced elevation in AST/ALT activity, the sinusoidal perfusion failure, and the number of TUNEL-positive hepatocytes were comparable between ESAM+/+ and ESAM−/− mice. Conclusions ESAM is expressed in the postischemic liver and mediates neutrophil but not T-cell transmigration during early reperfusion. ESAM deficiency attenuates I/R-induced vascular leakage and does not affect leukocyte adherence. Despite the effect on neutrophil migration, ESAM-deficiency does not protect from I/R-induced injury.

We have shown recently that vascular endothelial protein tyrosine phosphatase (VE-PTP), an endothelial-specific membrane protein, associates with vascular endothelial (VE)–cadherin and enhances VE-cadherin function in transfected cells (Nawroth, R., G. Poell, A. Ranft, U. Samulowitz, G. Fachinger, M. Golding, D.T. Shima, U. Deutsch, and D. Vestweber. 2002. EMBO J. 21:4885–4895). We show that VE-PTP is indeed required for endothelial cell contact integrity, because down-regulation of its expression enhanced endothelial cell permeability, augmented leukocyte transmigration, and inhibited VE-cadherin–mediated adhesion. Binding of neutrophils as well as lymphocytes to endothelial cells triggered rapid (5 min) dissociation of VE-PTP from VE-cadherin. This dissociation was only seen with tumor necrosis factor α–activated, but not resting, endothelial cells. Besides leukocytes, vascular endothelial growth factor also rapidly dissociated VE-PTP from VE-cadherin, indicative of a more general role of VE-PTP in the regulation of endothelial cell contacts. Dissociation of VE-PTP and VE-cadherin in endothelial cells was accompanied by tyrosine phoshorylation of VE-cadherin, β-catenin, and plakoglobin. Surprisingly, only plakoglobin but not β-catenin was necessary for VE-PTP to support VE-cadherin adhesion in endothelial cells. In addition, inhibiting the expression of VE-PTP preferentially increased tyrosine phosphorylation of plakoglobin but not β-catenin. In conclusion, leukocytes interacting with endothelial cells rapidly dissociate VE-PTP from VE-cadherin, weakening endothelial cell contacts via a mechanism that requires plakoglobin but not β-catenin.

Rationale: Endothelial cell–specific molecule 1 (Esm1) is a secreted protein thought to play a role in angiogenesis and inflammation. However, there is currently no direct in vivo evidence supporting a function of Esm1 in either of these processes. Objective: To determine the role of Esm1 in vivo and the underlying molecular mechanisms. Methods and Results: We generated and analyzed Esm1 knockout ( Esm1 KO ) mice to study its role in angiogenesis and inflammation. Esm1 expression is induced by the vascular endothelial growth factor A (VEGF-A) in endothelial tip cells of the mouse retina. Esm1 KO mice showed delayed vascular outgrowth and reduced filopodia extension, which are both VEGF-A–dependent processes. Impairment of Esm1 function led to a decrease in phosphorylated Erk1/2 (extracellular-signal regulated kinases 1/2) in sprouting vessels. We also found that Esm1 KO mice displayed a 40% decrease in leukocyte transmigration. Moreover, VEGF-induced vascular permeability was decreased by 30% in Esm1 KO mice and specifically on stimulation with VEGF-A 165 but not VEGF-A 121 . Accordingly, cerebral edema attributable to ischemic stroke–induced vascular permeability was reduced by 50% in the absence of Esm1. Mechanistically, we show that Esm1 binds directly to fibronectin and thereby displaces fibronectin-bound VEGF-A 165 leading to increased bioavailability of VEGF-A 165 and subsequently enhanced levels of VEGF-A signaling. Conclusions: Esm1 is simultaneously a target and modulator of VEGF signaling in endothelial cells, playing a role in angiogenesis, inflammation, and vascular permeability, which might be of potential interest for therapeutic applications.

During mouse preimplantation development, the components of the E-cadherin-catenin complex are derived from both maternal and zygotic gene activity and the adhesion complex is increasingly accumulated and stored in a nonfunctional form, ready to be used for compaction and the formation of the trophectoderm cell layer (Ohsugi et al., Dev. Dyn. 206:391–402, 1996). Here, we show that β-catenin is a major tyrosine-phosphorylated protein in oocytes and early cleavage-stage embryos and that the relative amount of phosphorylated β-catenin is greatly reduced during the morula-blastocyst transition. Peptide-specific antibodies indicate that β-catenin undergoes conformational changes and/or that the carboxy-terminal region of β-catenin is blocked during preimplantation development. Moreover, the availability of a carboxy-terminal epitope seems to depend on the tyrosine phosphorylation state of β-catenin and becomes unmasked when oocytes are treated with the tyrosine kinase inhibitor genistein. Our results suggest that tyrosine phosphorylation of β-catenin represents a molecular mechanism to keep the accumulating E-cadherin adhesion complex in a nonfunctional form. Dev Dyn 1999;216:168–176. © 1999 Wiley-Liss, Inc.

We have studied transcription, expression, and membrane localization of components of the E-cadherin-catenin complex stage by stage during mouse preimplantation development. Maternal E-cadherin and α- and β-catenin are stored as mRNA and/or protein in unfertilized eggs and are already assembled into a protein complex at this stage. After fertilization, it is likely that they mediate adhesion of early-stage blastomeres. Biosynthesis of plakoglobin is delayed relative to the other components. The temporal mRNA and protein expression patterns of the components of the cadherin-catenin complex correlate with the presence or absence of potential cytoplasmic polyadenylation elements (CPEs) in the 3′-UTRs of the respective cDNAs. Our results suggest that the components of the E-cadherin-catenin complex derived from both maternal and zygotic gene activity are increasingly accumulated and stored in a nonfunctional form during early cleavage stages and are ready to be used for compaction and the formation of the trophectodermal cell layer. © 1996 Wiley-Liss, Inc.

Classical cadherins are cell-surface glycoproteins that mediate calcium-dependent cell adhesion. The cytoplasmic domain of these glycoproteins is linked to the cytoskeleton through the catenins (alpha, beta and gamma). The catenins are intracellular polypeptides that are part of a complex sub-membranous network modulating the adhesive ability of the cells. One approach to elucidate the role of these molecules in the cell is to investigate their distribution during mouse development and in adult tissues. This study reports that catenins are widely expressed but in varying amounts in embryos and adult tissues. The expression of all three catenins is most prominent in the adult heart muscle and in epithelia of all developmental stages. In other embryonic and adult tissues, lower expression of catenins was detected, e.g., in smooth muscle or connective tissue. Catenins are coexpressed with various cadherins in different tissues. Gastrulation is the first time during embryogenesis when a discrepancy occurs between the expression of catenins and E-cadherin. E-cadherin expression is suppressed in mesodermal cells but not the expression of catenins. This discrepancy suggests that another cadherin may interact with catenins. Similarly, E-cadherin is generally expressed in adult liver but not in the regions surrounding the central veins. In contrast, catenins are uniformly expressed in the liver, suggesting that they are associated with other cadherins in E-cadherin negative cells. Finally, the three catenins are not always concurrently expressed. For example, in peripheral nerves, only beta-catenin is observable, and in smooth muscle plakoglobin is not detectable.

Background and objectives The LIM-and-SH3-domain-protein-1 (Lasp-1) is an actin-associated protein and is localised at focal adhesion sites where it is involved in organisation of actin polymerization and focal adhesion turnover prozesses. We investigated its role in regulating synovial fibroblast (SF) interaction with components of the extracellular matrix (ECM) and in establishing cell-cell contacts during RA. Materials and methods Lasp-1 expression was analysed in tissue from RA patients and in the hind paws of arthritic hTNFtg mice by Western blot, immunofluorescence and immunohistochemistry. Furthermore, Lasp-1-/- mice were interbred with hTNFtg mice and offsprings were analysed for the progression of joint destruction by clinical evaluation and histopathology. Migration characteristics of SF derived from wild type (wt), Lasp-1-/-, hTNFtg and Lasp1-/-/hTNFtg mice were analysed in a modified scratch assay and by live cell imaging. Cell-matrix interactions and cell-cell contacts of isolated SF from all different genotypes were investigated using fibronectin coating in an electrical cell/substrate impedance sensing assay (ECIS). Additionally, we used an in vitro three-dimensional organ culture system for functional analyes. Results Lasp-1 expression levels were increased in human RA tissue and hTNFtg mice in comparison to healthy controls. Evaluation of Lasp-1-/-/hTNFtg mice revealed milder arthritis score compared to hTNFtg mice,which was confirmed by immunohistochemistry. Results of the scratch assays demonstrated a significantly reduced migration rate of Lasp-1-/- SF (-43,7% vs wt SF) and Lasp-1-/-/hTNFtg SF (-69,11% vs hTNFtg SF). Furthermore, live cell imaging studies demonstrated striking differences in the migration velocity and in migration edge formation of Lasp-1-/-/hTNFtg SF compared to hTNFtg SF. ECIS analysis demonstrated an increased cell-cell contact formation in Lasp1-/- compared to wt SF (+22% versus wt SF) and prolonged cell-cell interaction remodelling of Lasp-1-/-/hTNFtg SF in comparison to hTNFtg SF. Histological analysis of 3D matrices showed that Lasp-1 deletion in the hTNFtg background resulted in an organised cellular lining layer at the interface between the matrix and fluid phases comparable with wild type SF. In contrast, hTNFtg SF formed unorganised cellular condensations with no synovial architecture evident. Conclusion Lasp-1 regulates the migratory behaviour of synovial fibroblasts in rheumatoid arthritis by controlling the dynamics of cell-matrix and cell-cell contacts.

The endothelium actively participates in neutrophil migration out of the vasculature via dynamic, cytoskeleton-dependent rearrangements leading to the formation of transmigratory cups in vitro, and to domes that completely surround the leukocyte in vivo. Leukocyte-specific protein 1 (LSP1), an F-actin–binding protein recently shown to be in the endothelium, is critical for effective transmigration, although the mechanism has remained elusive. Herein we show that endothelial LSP1 is expressed in the nucleus and cytosol of resting endothelial cells and associates with the cytoskeleton upon endothelial activation. Two-photon microscopy revealed that endothelial LSP1 was crucial for the formation of endothelial domes in vivo in response to neutrophil chemokine keratinocyte-derived chemokine (KC) as well as in response to endogenously produced chemokines stimulated by cytokines (tumor necrosis factor α [TNFα] or interleukin-1β [IL-1β]). Endothelial domes were significantly reduced in Lsp1−/− compared with wild-type (WT) mice. Lsp1−/− animals not only showed impaired neutrophil emigration after KC and TNFα stimulation, but also had disproportionate increases in vascular permeability. We demonstrate that endothelial LSP1 is recruited to the cytoskeleton in inflammation and plays an important role in forming endothelial domes thereby regulating neutrophil transendothelial migration. The permeability data may underscore the physiologic relevance of domes and the role for LSP1 in endothelial barrier integrity.

Leukocyte extravasation depends on various adhesion receptors at endothelial cell contacts. Here we have analyzed how mouse CD99 and CD99L2 cooperate with PECAM-1. We found that antibodies against mouse CD99 and PECAM-1 trap neutrophils between endothelial cells in in vitro transmigration assays. A sequential function, as has been suggested for human PECAM-1 and CD99, could not be demonstrated. In contrast to these in vitro results, blocking CD99 or CD99L2 or gene disruption of PECAM-1 trapped neutrophils in vivo between endothelial cells and the underlying basement membrane as revealed by electron microscopy and by 3-dimensional confocal fluorescence microscopy in the inflamed cremaster tissue. Leukocyte extravasation was inhibited in interleukin-1β-inflamed peritoneum and in the cremaster by PECAM-1 gene disruption and was further attenuated by blocking antibodies against CD99 and CD99L2. In addition, CD99 and CD99L2 were required for leukocyte extravasation in the cremaster after stimulation with tumor necrosis factor-α, where the need for PECAM-1 is known to be bypassed. We conclude that CD99 and CD99L2 act independently of PECAM-1 in leukocyte extravasation and cooperate in an independent way to help neutrophils overcome the endothelial basement membrane.

Hematopoietic stem cells (HSCs) have been highly enriched using combinations of 12–14 surface markers. Genes specifically expressed by HSCs as compared with other multipotent progenitors may yield new stem cell enrichment markers, as well as elucidate self-renewal and differentiation mechanisms. We previously reported that multiple cell surface molecules are enriched on mouse HSCs compared with more differentiated progeny. Here, we present a definitive expression profile of the cell adhesion molecule endothelial cell-selective adhesion molecule (Esam1) in hematopoietic cells using reverse transcription-quantitative polymerase chain reaction and flow cytometry studies. We found Esam1 to be highly and selectively expressed by HSCs from mouse bone marrow (BM). Esam1 was also a viable positive HSC marker in fetal, young, and aged mice, as well as in mice of several different strains. In addition, we found robust levels of Esam1 transcripts in purified human HSCs. Esam1−/− mice do not exhibit severe hematopoietic defects; however, Esam1−/− BM has a greater frequency of HSCs and fewer T cells. HSCs from Esam1−/− mice give rise to more granulocyte/monocytes in culture and a higher T cell:B cell ratio upon transplantation into congenic mice. These studies identify Esam1 as a novel, widely applicable HSC-selective marker and suggest that Esam1 may play roles in both HSC proliferation and lineage decisions.

Although recent advances have enabled hematopoietic stem cells (HSCs) to be enriched to near purity, more information about their characteristics will improve our understanding of their development and stage-related functions. Here, using microarray technology, we identified endothelial cell-selective adhesion molecule (ESAM) as a novel marker for murine HSCs in fetal liver. Esam was expressed at high levels within a Rag1− c-kitHi Sca1+ HSC-enriched fraction, but sharply down-regulated with activation of the Rag1 locus, a valid marker for the most primitive lymphoid progenitors in E14.5 liver. The HSC-enriched fraction could be subdivided into 2 on the basis of ESAM levels. Among endothelial antigens on hematopoietic progenitors, ESAM expression showed intimate correlation with HSC activity. The ESAMHi population was highly enriched for multipotent myeloid-erythroid progenitors and primitive progenitors with lymphopoietic activity, and exclusively reconstituted long-term lymphohematopoiesis in lethally irradiated recipients. Tie2+ c-kit+ lymphohematopoietic cells in the E9.5–10.5 aorta-gonad-mesonephros region also expressed high levels of ESAM. Furthermore, ESAM was detected on primitive hematopoietic progenitors in adult bone marrow. Interestingly, ESAM expression in the HSC-enriched fraction was up-regulated in aged mice. We conclude that ESAM marks HSC in murine fetal liver and will facilitate studies of hematopoiesis throughout life.

Invasion of lymphatic vessels is a key step in the metastasis of primary tumors to draining lymph nodes. Although the process is enhanced by tumor lymphangiogenesis, it is unclear whether this is a consequence of increased lymphatic vessel number, altered lymphatic vessel properties, or both. Here we have addressed the question by comparing the RNA profiles of primary lymphatic endothelial cells (LEC) isolated from the vasculature of normal tissue and from highly metastatic T-241/vascular endothelial growth factor (VEGF)-C fibrosarcomas implanted in C57BL/6 mice. Our findings reveal significant differences in expression of some 792 genes (i.e., ≥2-fold up- or down-regulated, P ≤ 0.05) that code for a variety of proteins including components of endothelial junctions, subendothelial matrix, and vessel growth/patterning. The tumor LEC profile, validated by immunohistochemical staining, is distinct from that of normal, inflammatory cytokine, or mitogen-activated LEC, characterized by elevated expression of such functionally significant molecules as the tight junction regulatory protein endothelial specific adhesion molecule (ESAM), the transforming growth factor-β coreceptor Endoglin (CD105), the angiogenesis-associated leptin receptor, and the immunoinhibitory receptor CD200, and reduced expression of subendothelial matrix proteins including collagens, fibrillin, and biglycan. Moreover, we show similar induction of ESAM, Endoglin, and leptin receptor within tumor lymphatics in a series of human head and neck and colorectal carcinomas, and uncover a dramatic correlation between ESAM expression and nodal metastasis that identifies this marker as a possible prognostic indicator. These findings reveal a remarkable degree of phenotypic plasticity in cancer lymphatics and provide new insight into the processes of lymphatic invasion and lymph node metastasis. [Cancer Res 2008;68(18):7293–303]

The cover shows a double immunofluorescence staining of ESAM-1 and PECAM-1 in the T cell zone of the peripheral lymph node of the mouse. Red immunofluorescence of ESAM-1 colocalizes with the bright green immunofluorescence of PECAM-1, resulting in the observed yellow immunofluorescence. Lymphatic endothelial cells express PECAM-1 but lack ESAM-1 and hence are manifested as dim green immunofluorescence. The image was taken from the article by Pfeiffer et al. ( pp. 2142–2155) in which the authors demonstrate that molecularly distinct cellular junctions of blood and lymphatic endothelial cells establish different functional barriers controlling the distribution antigens and immune cells in the peripheral lymph node.

Recirculation of fluid and cells through lymphatic vessels plays a key role in normal tissue homeostasis, inflammatory diseases, and cancer. Despite recent advances in understanding lymphatic function (Alitalo, K., T. Tammela, and T.V. Petrova. 2005. Nature. 438:946–953), the cellular features responsible for entry of fluid and cells into lymphatics are incompletely understood. We report the presence of novel junctions between endothelial cells of initial lymphatics at likely sites of fluid entry. Overlapping flaps at borders of oak leaf–shaped endothelial cells of initial lymphatics lacked junctions at the tip but were anchored on the sides by discontinuous button-like junctions (buttons) that differed from conventional, continuous, zipper-like junctions (zippers) in collecting lymphatics and blood vessels. However, both buttons and zippers were composed of vascular endothelial cadherin (VE-cadherin) and tight junction–associated proteins, including occludin, claudin-5, zonula occludens–1, junctional adhesion molecule–A, and endothelial cell–selective adhesion molecule. In C57BL/6 mice, VE-cadherin was required for maintenance of junctional integrity, but platelet/endothelial cell adhesion molecule–1 was not. Growing tips of lymphatic sprouts had zippers, not buttons, suggesting that buttons are specialized junctions rather than immature ones. Our findings suggest that fluid enters throughout initial lymphatics via openings between buttons, which open and close without disrupting junctional integrity, but most leukocytes enter the proximal half of initial lymphatics.

Leukocyte emigration into inflamed tissues is among the most intensely pursued topics in the field of inflammation. Research focuses on the molecular factors activating endothelial cells and leukocytes, the adhesive molecules facilitating the contact between both cell types and the mechanisms allowing leukocytes to transmigrate through the blood vessel endothelium. In the last few years, studies have been intensified to understand how leukocytes, once captured to the vessel wall overcome the barrier made of endothelial cells linked to each other by interendothelial junctions. The mechanisms by which these leukocytes traverse the endothelial cell layer to reach the underlying tissue, a process called diapedesis, are largely unknown. Whereas convincing evidence has been published that polymorphonuclear leukocytes (PMN) can indeed migrate through endothelial cells in a transcellular fashion in vivo [1] as well as in vitro [2], careful quantitative analysis has demonstrated that at least in vitro the majority of PMNs and other leukocytes migrate via a paracellular route through the contact areas between endothelial cells [2, 3]. Consequently, a number of endothelial cell contact proteins such as PECAM-1, members of the junctional adhesion molecule family (JAM-A, -B and -C), CD99 and ICAM-2 have been reported to support leukocyte extravasation [4, 5, 6, 7] . PECAM-1 was the first of these proteins that was identified in the context of leukocyte extravasation [8]. Its relevance for neutrophil extravasation is well established [9]. Although PECAM-1, JAM-A and ICAM-2 were shown by intravital microscopy to be involved in the transmigration process in vivo, the detailed molecular mechanism by which they participate in the process is still unknown.

Background and objectives Lasp-1 (LIM-and-SH3-domain-protein-1) is an actin-binding protein that modifies cytoskeleton organisation and is localised at cell-matrix interaction sites where it is involved in cell adhesion, migration and metastatic invasion. Its role in regulating synovial fibroblast (SF) interaction with components of extracellular matrix (ECM) and cell-cell contacts during RA is unknown. Furthermore, involved signalling pathways have to be elucidated. Materials and methods Lasp-1 expression in RA tissue and hind paws of arthritic hTNFtg mice was analysed via Western blot and immunohistochemistry. Furthermore, Lasp-1 ko mice were interbred with hTNFtg mice and offsprings were analysed for the progression of joint destruction by clinical evaluation and histopathology. Cell migration properties of SF derived from wild type (wt), Lasp-1 -/- , hTNFtg and Lasp1 -/- /hTNFtg mice were measured using a modified scratch assay and by live cell imaging. Extracellular matrix structure and organisation from wt, Lasp-1 -/- , hTNFtg and Lasp-1 -/- /hTNFtg SF were analysed by electron microscopy (EM). Cell-matrix interactions as well as cell-cell contacts of isolated SF from mice of all genotypes were investigated using fibronectin and self-purified collagen matrix in an electrical cell/substrate impedance sensing assay (ECIS). Via pull down assays and EM components of integrin mediated cell-matrix interaction complexes were examined. In addition, src-phosphorylation levels were evaluated by Western blot. Results Lasp-1 expression levels were increased in human RA and in hTNFtg mice compared to healthy controls. Lasp-1 -/- /hTNFtg mice displayed milder clinical symptoms confirmed by immunohistochemistry. Migration assays presented a significantly reduced migration rate of Lasp-1 -/- and Lasp-1 -/- /hTNFtg SF compared to SF from wt and hTNFtg mice. Analyses of SF ECM showed thickened collagen fibrils with less crosslinking of Lasp-1 -/- SF in comparison to wt controls. In ECIS, Lasp-1 -/- SF adhered to both fibronectin and self purified ECM faster than other genotypes (-50% vs. hTNFtg and wt SF). Additionally, Lasp-1 -/- SF formed a higher number of cell-cell contacts than wt and hTNFtg SF (-17% vs. hTNFtg and -22% wt vs. SF). Pull down assays identified cortactin, uPar and vinculin as components of cell-matrix interaction complexes with increased levels of vinculin found in complexes from Lasp-1 -/- compared to wtSF. Finally, both Lasp-1 -/- and Lasp-1 -/- /hTNFtg SF showed reduced src-phosphorylation compared to wt and hTNFtg SF. Conclusion The loss of Lasp-1 leads to altered src-phosphorylation and changes in cell-matrix interaction complexes as well as altered extracellular matrix organisation. Furthermore, Lasp-1 deficiency leads to significantly reduced migration rates of RASF and hTNFtg SF in vitro and to decreased cartilage degradation in hTNFtg mice in vivo .

Immature dendritic cells (DC) reside in tissues where they initiate immune responses by taking up foreign antigens. Since DC have a limited tissue half-life, the DC pool in tissues has to be replenished constantly. This implies that precursor/immature DC must be able to cross non-activated endothelium using as yet unknown mechanisms. Here we show that immature, but not mature bone marrow-derived murine DC migrate across resting endothelial monolayers in vitro. We find that endothelial intercellular adhesion molecule-2 (ICAM-2) is a major player in transendothelial migration (TEM) of immature DC, accounting for at least 41% of TEM. Surprisingly, the ICAM-2-mediated TEM was independent of beta2-integrins, the known ICAM-2 ligands, since neither blocking of beta2-integrins with antibodies nor the use of CD18-deficient DC affected the ICAM-2-specific TEM. In humans, the C-type lectin DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) was shown to interact with ICAM-2, suggesting a similar role in mice. However, we find that none of the murine DC-SIGN homologues mDC-SIGN, murine DC-SIGN-related molecule-1 (mSIGN-R1) and mSIGN-R3 is expressed on the surface of bone marrow-derived mouse DC. Taken together, this study shows that ICAM-2 strongly supports transmigration of immature DC across resting endothelium by interacting with ligands that are distinct from beta2-integrins and DC-SIGN homologues.

Human CD99 is a small highly O-glycosylated cell-surface protein expressed on most leukocytes. It was recently found to be expressed at endothelial cell contacts and to participate in the transendothelial migration (TEM) of monocytes in vitro. In order to analyze the physiologic relevance of CD99 in vivo we searched for the mouse homolog. We cloned a mouse cDNA coding for a protein 45% identical in its sequence with human CD99. Based on the cDNA, we generated antibodies against this mouse homolog of CD99, which detected the antigen on most leukocytes, on endothelia of various tissues, and at cell contacts of cultured endothelial cells. Cell aggregation of CD99-transfected Chinese hamster ovary (CHO) cells was completely blocked by anti-CD99 antibodies. The same antibodies inhibited TEM of lymphocytes in vitro, independent of whether T cells or endothelial cells were preincubated with antibodies. In a cutaneous delayed-type hypersensitivity (DTH) reaction, anti-CD99 antibodies inhibited the recruitment of in vivo–activated T cells into inflamed skin as well as edema formation. We conclude that mouse CD99 participates in the TEM of lymphocytes and in their recruitment to inflamed skin in vivo. This establishes CD99 as a valid target for interference with cutaneous inflammatory processes.

Background and Objectives Lasp-1 is an important actin-crosslinking protein. It’s localises at focal adhesions, along stress fibres and leading edges and is involved in cellular migration and metastatic dissemination of different cancers. Rheumatoid arthritis synovial fibroblasts (RASF) are also able to enter the bloodstream from sites of cartilage destruction to new locations where they re-initiate the destructive processes. The underlying mechanisms of this process are of special interest, but currently unclear. Therefore we have investigated the role of Lasp-1 in synovial fibroblast migration during RA. Materials and Methods Lasp-1 expression and its subcellular location was analysed using Western blotting and immunofluorescence microscopy of cells seeded on various coatings, including fibronectin, laminin and a self-purified collagen matrix. Furthermore, Lasp-1 deficient mice were generated, which were interbred with hTNFtg mice, to form a model of inflammatory arthritis. Mice of all genotypes were analysed using a clinical score, measuring weight, grip strength and paw swelling over a time course of 14 weeks. Hind paws from each genotype were isolated, paraffin-embedded and toluidine blue stained in order to assess synovial inflammation, cartilage degradation and SF attachment. Cell migration properties of SFs derived from WT, Lasp-1-/-, hTNFtg and Lasp1-/-/hTNFtg were measured using a modified scratch assay and by live cell imaging. Results Lasp-1 expression was up regulated in RASF and in SF from hTNFtg mice compared to healthy controls. In immunofluorescence, Lasp-1 was co-localised with cortactin, a marker for structures of cell adhesion and invasion, particularly when cultured on a purified collagen matrix. Lasp1-/-/hTNFtg mice showed milder clinical symptoms in comparison to hTNFtg mice. Notably, histopathological analyses revealed less cartilage destruction (0.4 mm vs. 1.6 mm, p Conclusion Lasp-1 deficiency leads to significantly reduced migration rates of RASF and hTNFtg SF in vitro and results in a decreased cartilage degradation and destruction and less SF attachment to articular cartilage in hTNFtg mice in vivo. Therefore, Lasp-1 is an important target for therapeutic strategies aimed to reduce the invasive and migratory behaviour of synovial fibroblast in rheumatoid arthritis.

Catenins are proteins associated with the cytoplasmic domain of cadherins, a family of transmembrane cell adhesion molecules. The cadherin–catenin adhesion system is involved in morphogenesis during development and in the maintenance of the integrity of different tissue types. Using a gene trap strategy, we have isolated a mouse mutation for the gene encoding the α-E-catenin. This form of the α-catenin appears frequently coexpressed with E-cadherin in epithelial cell types. The mutation obtained eliminates the carboxyl-terminal third of the protein but nevertheless provokes a complete loss-of-function phenotype. Homozygous mutants show disruption of the trophoblast epithelium (the first differentiated embryonic tissue), and development is consequently blocked at the blastocyst stage. This phenotype parallels the defects observed in E-cadherin mutant embryos. Our results show the requirement of the α-E-catenin carboxy terminus for its function and represent evidence of the role of the α-E-catenin in vivo , identifying this molecule as the natural partner of the E-cadherin in trophoblast epithelium.

Background Recent studies demonstrated the potential of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) to emigrate from affected joints and migrate via the bloodstream toward distant healthy cartilage in the SCID mouse model of disease. While the mechanisms of breaking endothelial barriers by RA-FLS are largely unclear, the junctional adhesion molecule C (JAM-C) has become of interest in this context due to its involvement in trans-endothelial migration of leukocytes. Using the human TNF transgenic (hTNFtg) mouse as a model for human RA, we studied the role of JAM-C in the transmigration of FLS derived from these mice. Methods The expression pattern of JAM-C on wild-type and hTNFtg FLS was analyzed by Western-blot and immunocytochemistry. We investigated the transmigratory capacity of these cells in a transmigration assay using murine endothelioma cells (bEnd.5) as an endothelial barrier, IL-1alpha treated murine cartilage tissue served as chemoattractant stimulus within this setting. Functional analyses included the knock down of JAM-C expression by siRNA against murine JAM-C as well as its neutralization by blocking antibodies. Results The expression of JAM-C could be detected on the surface of both wild-type and hTNFtg FLS and it was primarly located on sites of cell-cell interactions. Additionally, Western blot data showed an elevated expression of JAM-C in FLS from hTNFtg mice. Our transmigration experiments demonstrated a markedly higher potential of hTNFtg FLS to migrate through the endothelial monolayer than FLS from wild-type mice (+40%, p≤0.05), and cartilage explants pre-treated with IL-1alpha as chemoattractant strengthened the migratory capacity of hTNFtg FLS. Interestingly, knock down of JAM-C expression on hTNFtg FLS mediated by siRNA decreased the number of transmigrated cells of about 40% comparing with mock control (p≤0.01). Equally, the neutralization of JAM-C by blocking antibodies against murine JAM-C resulted in a reduction of transmigration on a similar level. Conclusions The inflammatory environment characteristic for joints of hTNFtg mice induces an up-regulation of JAM-C on FLS similar to that of human FLS during RA. Moreover, the differentiation of a trans-migrating phenotype of FLS, capable to break through endothelial barriers is supported by this environment. In this context, JAM-C seems to be contributed to this process of transmigration and, thus, to the extravasation of FLS and, therefore targeting JAM-C may be a promising therapeutic strategy for RA. Disclosure of Interest None Declared

Background and Objectives RA synovial fibroblasts (SF) have been suggested to contribute to the spreading of disease through their ability to leave cartilage destruction sites, migrate via the bloodstream and re-initiate the destructive process at distant articular cartilage surfaces. In this context, the actin-crosslinking protein Lasp-1 is of interest, because it is localised at leading edges of migrating cells and regulates metastatic dissemination of different tumours. Therefore, it is particularly important to investigate the role of Lasp-1 in SF migration and its effects on RA. Materials and Methods To identify different Lasp-1 expression levels in the hind paws of wt and hTNFtg mice, an established model for human RA, Western- blot analyses were performed. In parallel, Lasp-1 expression and its sub-cellular distribution was investigated in SF from wt and hTNFtg mice by Western-blot analyses and immunofluorescence. The migratory capacity of SFs derived from wild-type, Lasp-1 -/- , hTNFtg and Lasp1 -/- /hTNFtg mice was studied in a modified scratch assay as well as in live cell imaging studies. Furthermore, a transmigration assay using SF from all four genotypes and murine endothelioma cells (bEnd.5) as an endothelial barrier was carried out. For more detailed information, SF transmigration was evaluated when endothelial cells were also pre-treated with TNF-alpha, mimicking inflammatory conditions. Results Lasp-1 expression is upregulated in SF from hTNFtg mice and localises to structures of cell adhesion and invasion. In the scratch assay, a significantly reduced migration rate was detected in Lasp-1 -/- SFs after 24 hrs (–43.7% versus wt, p -/- /hTNFtg, respectively (–69.11% versus hTNFtg, p -/- /hTNFtg compared to hTNFtg SF. Furthermore, analyses showed a significant reduction of transmigration of Lasp1 -/- /hTNFtg compared to hTNFtg SF that was even enhanced by TNF-alpha stimulation of the endothelial cells. Interestingly, interbred Lasp1 -/- /hTNFtg mice presented milder clinical symptoms and analyses of histopathology revealed less cartilage degradation and less attachment of synovial tissue to the cartilage than hTNFtg mice at an age of 14 weeks. Conclusions Our data provide that the migratory capacity of SF is regulated by Lasp-1 and influences the severity of arthritis in hTNFtg mice. SF – when activated – migrate through the formation of invasive and adhesive membrane structures such as invadopodia, where Lasp-1 is prominently localised. Thus, targeting Lasp-1 may be a promising strategy to reduce the invasive and migratory behaviour of synovial fibroblasts in RA.

Background and objectives Recent studies demonstrated the potential of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) to migrate long distances via the bloodstream and invade distant cartilage in the SCID mouse model of disease.While the mechanisms of trans-endothelial migration of RA-FLS are largely unclear the junctional adhesion molecule C (JAM-C) has become of interest due to its involvement in trans-endothelial migration of leucocytes. Here, the authors used the hTNFtg mouse as a model for human RA and studied the role of JAM-C in the transmigration of FLS derived from these mice. Materials and methods The expression of JAM-C on wild-type and hTNFtg FLS was investigated by western-blot analysis and immunocytochemistry. The transmigratory capacity of these cells was studied in a transmigration assay using murine endothelioma cells (bEnd.5) as an endothelial barrier and IL-1alpha treated murine cartilage tissue as chemoattractant stimulus. Functional analyses included the knock down of JAM-C expression by siRNA against murine JAM-C as well as its neutralisation by blocking antibodies. Results The authors found the expression of JAM-C on the surface of both wild-type and hTNFtg FLS which is prominently located on sites of cell-cell interactions. Moreover, Western blot data revealed an elevated expression of JAM-C in FLS from hTNFtg mice. Transmigration experiments demonstrated a significantly higher potential of hTNFtg FLS to migrate through the endothelial monolayer than FLS from wild-type mice (+40%, p≤0,05), and cartilage explants pretreated with IL-1α enhanced the migratory capacity of hTNFtg FLS. Interestingly, siRNA-mediated knock down of JAM-C expression on hTNFtg FLS resulted in a reduction of transmigration of about 40% compared to mock control (p≤0,01). Likewise, the neutralisation of JAM-C by blocking antibodies against murine JAM-C reduced the number of transmigrated hTNFtg FLS on a similar level. Conclusion Our data demonstrate that the inflammatory environment within the joints of hTNFtg mice induces an up-regulation of JAM-C on FLS, which is characteristic for human RA-FLS. Moreover, they indicate that this environment supports the development of a trans-migrating phenotype of FLS able to get through endothelial barriers. JAM-C seems to be involved functionally in the transmigration and, thus, in extravasation of FLS and, therefore targeting JAM-C may be a promising therapeutic strategy for RA.

In the course of an analysis of cell-cell adhesion in the Xenopus embryo, antibodies directed against α and β catenin were applied to investigate their relation to the cadherins occurring early in this system. The results demonstrate that α and β-catenin are provided maternally and increase in amount throughout embryogenesis. Immunoprecipitations indicate that both of the catenins are complexed to U-cadherin in the early phase of embryogenesis and to β-cadherin, when it appears during gastrulation. An excess of α-catenin occurs in free form in the early embryo, whereas all of the catenin seems to be complexed to cadherin. Synthesis of the two components throughout early embryogenesis and their binding to newly synthesized cadherins were demonstrated by metabolic labelling. The spatial distribution of α-catenin was analysed by immunohistology. During cleavage β-catenin is deposited evenly along the plasma membranes within the embryo, while the cell peripheries at the surface of the embryo remain devoid of α-catenin. At later stages, the pattern of α-catenin distribution becomes more complex. Quantitative differences in the intensity of staining along the plasma membranes in the different regions of the embryo can be distinguished. Particularly the appearance of β-cadherin in the gastrula ectoderm is accompanied by conspicous depositions of α-catenin along the respective plasma membranes in this layer. All cells in the later embryo, apart from the neural crest cells, carry α-catenin on their plasma membranes indicating the universal character of cadherin-mediated cell-cell adhesion in the Xenopus embryo.

One of the distinct biochemical signatures of life is the high chiral preference of biomolecules that compose the organisms, e.g.L-amino acids, D-sugars, L-phospholipids. As a result, many biological and physiological processes are greatly influenced by the chirality of biomolecules. This inspires the introduction of chiral effects into biomaterials research. For this purpose, we developed a novel chiral polymer brush film system containing amino acid units and report that the stereochemistry of the polymer films significantly influences the cell/substrate interaction. With two adhesive cell lines—COS-7 and bEnd.3 as examples, here we report a study of differential cell behaviors on chiral polymer film. We show that the cells can adhere, grow, spread, and assemble much better on the L-amino acid based polymer film than on the corresponding D film, although their chemical and other physical properties are all the same, indicating that the L configuration of the polymer film has better cytocompatibility than the D configuration. It not only implies a novel strategy for the design of new generation of biomaterials and devices based on the chiral effect, but may also add important knowledge to the understanding the origin of chiral preference in biosystems.

Hematopoietic stem cells (HSC) are an important cell type with the capacity for self-renewal as well as differentiation into multi-lineage blood cells, maintaining the immune system throughout life. Many studies have attempted to identify unique markers associated with these extremely rare cells. In bone marrow of adult mice, the Lin-c-kitHi Sca1+ CD34−/Lo Thy1.1Lo subset is known to include HSC with long-term repopulating capacity. However, several of these parameters differ between strains of mice, change dramatically during developmental age and/or are expressed on many non-HSC during inflammation. Efficient HSC-based therapies and the emerging field of regenerative medicine will benefit from learning more about what defines stem cells. We previously determined that the most primitive cells with lymphopoietic potential first develop in the paraaortic splanchnopleura/aorta-gonad-mesonephros (AGM) region of embryos using Rag1/GFP knock-in mice. We also reported that Rag1/GFP-c-kitHi Sca1+ cells derived from E14.5 fetal liver (FL) reconstituted lympho-hematopoiesis in lethally irradiated adults, while Rag1/GFPLo c-kitHi Sca1+ cells transiently contributed to T and B lymphopoiesis. To extend those findings, microarray analyses were conducted to search for genes that characterize the initial transition of fetal HSC to primitive lymphopoietic cells. The comparisons involved mRNA from Rag1Lo ckitHi Sca1+, early lymphoid progenitors (ELP) and the HSC-enriched Rag1-ckitHi Sca1+ fraction isolated from E14.5 FL. While genes potentially related to early lymphopoiesis were discovered, our screen also identified genes whose expression seemed to correlate with HSC. Among those, endothelial cell-selective adhesion molecule (ESAM) attracted attention because of its conspicuous expression in the HSC fraction and sharp down-regulation on differentiation to ELP. ESAM was originally identified as an endothelial cell-specific protein, but expression on megakaryocytes and platelets was also reported (J. Biol. Chem., 2001, 2002). Flow cytometry analyses with anti-ESAM antibodies showed that the HSC-enriched Rag1-c-kitHi Sca1+ fraction could be subdivided into two on the basis of ESAM levels. The subpopulation with the high density of ESAM was enriched for c-kitHi Sca1Hi cells, while ones with negative or low levels of ESAM were found in the c-kitHi Sca1Lo subset. Among endothelial-related antigens on HSC, CD34 and CD31/PECAM1 were uniformly present on Rag1-c-kitHi Sca1+ cells in E14.5 FL and neither resolved into ESAMHi and ESAM−/Lo fractions. Expression profiles of Endoglin and Tie2 partially correlate with ESAM. The primitive ESAMHi fraction uniformly expressed high levels of Endoglin and Tie2, but many of the more differentiated ESAM−/Lo cells still retained the two markers. ESAM expression correlated well with HSC activity. Cells in the ESAMHi Rag1-ckitHi Sca1+ fraction formed more and larger colonies than those in the ESAM-/Lo Rag1-ckitHi Sca1+ fraction. Particularly, most CFU-Mix, primitive progenitors with both myeloid and erythroid potential, were found in the ESAMHi fraction. In limiting dilution stromal cell co-cultures, we found that 1 in 2.1 ESAMHi Rag1-ckitHi Sca1+ cells and 1 in 3.5 ESAM−/Lo Rag1-ckitHi Sca1+ cells gave rise to blood cells. However, while only 1 in 125 ESAM−/Lo Rag1-ckitHi Sca1+ cells were lymphopoietic under these conditions, 1 in 8 ESAMHi Rag1-ckitHi Sca1+ cells produced CD19+ B lineage cells. In long-term reconstituting assays, ESAMHi Rag1-ckitHi Sca1+ cells contributed highly to the multi-lineage recovery of lympho-hematopoiesis in recipients, but no chimerism was detected in mice transplanted with ESAM−/Lo Rag1-ckitHi Sca1+ cells. These results suggested that HSC in E14.5 FL are exclusively present in the ESAMHi fraction. Tie2+ c-kit+ lympho-hematopoietic cells of E10.5 AGM also expressed high levels of ESAM. Furthermore, ESAM expression in adult bone marrow was detected on primitive progenitors and cells in the side population within the Lin-ckitHi Sca1+ fraction. Interestingly, the expression was up-regulated in aged mice. Based on these observations, we conclude that ESAM marks HSC throughout life in mice. We also observed that many of human cord blood CD34+ CD38− cells express ESAM, suggesting potential application for the purification of human HSC.