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Supplementary Figures 1-8 from A Novel Gene Expression Profile in Lymphatics Associated with Tumor Growth and Nodal Metastasis

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]

Mammalian de novo DNA methyltransferases (DNMT) are responsible for the establishment of cell-type-specific DNA methylation in healthy and diseased tissues. Through genome-wide analysis of de novo methylation activity in murine stem cells we uncover that DNMT3A prefers to methylate CpGs followed by cytosines or thymines, while DNMT3B predominantly methylates CpGs followed by guanines or adenines. These signatures are further observed at non-CpG sites, resembling methylation context observed in specialised cell types, including neurons and oocytes. We further show that these preferences result from structural differences in the catalytic domains of the two de novo DNMTs and are not a consequence of differential recruitment to the genome. Molecular dynamics simulations suggest that, in case of human DNMT3A, the preference is due to favourable polar interactions between the flexible Arg836 side chain and the guanine that base-pairs with the cytosine following the CpG. By exchanging arginine to a lysine, the corresponding side chain in DNMT3B, the sequence preference is reversed, confirming the requirement for arginine at this position. This context-dependent enzymatic activity provides additional insights into the complex regulation of DNA methylation patterns.

Chromosomes have an intrinsic tendency to segregate into compartments, forming long‐distance contacts between loci of similar chromatin states. How genome compartmentalization is regulated remains elusive. Here, comparison of mouse ground‐state embryonic stem cells (ESCs) characterized by open and active chromatin, and advanced serum ESCs with a more closed and repressed genome, reveals distinct regulation of their genome organization due to differential dependency on BAZ2A/TIP5, a component of the chromatin remodeling complex NoRC. On ESC chromatin, BAZ2A interacts with SNF2H, DNA topoisomerase 2A (TOP2A) and cohesin. BAZ2A associates with chromatin sub‐domains within the active A compartment, which intersect through long‐range contacts. We found that ground‐state chromatin selectively requires BAZ2A to limit the invasion of active domains into repressive compartments. BAZ2A depletion increases chromatin accessibility at B compartments. Furthermore, BAZ2A regulates H3K27me3 genome occupancy in a TOP2A‐dependent manner. Finally, ground‐state ESCs require BAZ2A for growth, differentiation, and correct expression of developmental genes. Our results uncover the propensity of open chromatin domains to invade repressive domains, which is counteracted by chromatin remodeling to establish genome partitioning and preserve cell identity., The NoRC chromatin remodeller subunit BAZ2A restricts active chromatin compartments and secures developmental gene expression in ESCs.

Chromatin modifications regulate genome function by recruiting proteins to the genome. However, the protein composition at distinct chromatin modifications has yet to be fully characterized. In this study, we used natural protein domains as modular building blocks to develop engineered chromatin readers (eCRs) selective for DNA methylation and histone tri-methylation at H3K4, H3K9 and H3K27 residues. We first demonstrated their utility as selective chromatin binders in living cells by stably expressing eCRs in mouse embryonic stem cells and measuring their subnuclear localization, genomic distribution and histone-modification-binding preference. By fusing eCRs to the biotin ligase BASU, we established ChromID, a method for identifying the chromatin-dependent protein interactome on the basis of proximity biotinylation, and applied it to distinct chromatin modifications in mouse stem cells. Using a synthetic dual-modification reader, we also uncovered the protein composition at bivalently modified promoters marked by H3K4me3 and H3K27me3. These results highlight the ability of ChromID to obtain a detailed view of protein interaction networks on chromatin. The protein complexes associated with specific chromatin marks in living cells are identified using engineered binding proteins.

Chromosomes have an intrinsic tendency to segregate into compartments, forming long-distance contacts between loci of similar chromatin states. However, how genome compartmentalization is regulated remains elusive. We analyzed two closely and developmentally related pluripotent cell types: ground-state ESCs that have an open and active chromatin and developmentally advanced ESCs that display a more closed and repressed state. We show that these two ESC types differ in their regulation of genome organization due to their differential dependency on TIP5, a component of the chromatin remodeling complex NoRC. We show that TIP5 interacts on ESC chromatin with SNF2H, DNA topoisomerase 2A (TOP2A) and cohesin. TIP5 associates with sub-domains within the active A compartment that strongly intersect through long-range contacts in ESCs. We found that only ground-state chromatin requires TIP5 to limit the invasion of active domains into repressive compartments. Depletion of TIP5 increased chromatin accessibility particularly at B compartments and decreased their repressive features. Furthermore, TIP5 acts as a barrier for the repressive H3K27me3 spreading, a process that also requires TOP2A activity. Finally, ground-state ESCs require TIP5 for growth, differentiation capacity, and correct expression of developmental genes. Our results revealed the propensity of open and active chromatin domains to invade repressive domains, an action counteracted by chromatin remodeling and the relief of chromatin torsional stress. This effort in controlling open/active chromatin domains is required to establish active and repressed genome partitioning and preserves cell function and identity.

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.

Forward genetic screens with genome-wide CRISPR libraries are powerful tools for resolving cellular circuits and signaling pathways. Applying this technology to organoids, however, has been hampered by technical limitations. Here we report improved accuracy and robustness for pooled-library CRISPR screens by capturing sgRNA integrations in single organoids, substantially reducing required cell numbers for genome-scale screening. We applied our approach to wild-type and APC mutant human intestinal organoids to identify genes involved in resistance to TGF-β-mediated growth restriction, a key process during colorectal cancer progression, and validated hits including multiple subunits of the tumor-suppressive SWI/SNF chromatin remodeling complex. Mutations within these genes require concurrent inactivation of APC to promote TGF-β resistance and attenuate TGF-β target gene transcription. Our approach can be applied to a variety of assays and organoid types to facilitate biological discovery in primary 3D tissue models.

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.

Oct4, Sox2, Klf4, and cMyc (OSKM) reprogram somatic cells to pluripotency. To gain a mechanistic understanding of their function, we mapped OSKM-binding, stage-specific transcription factors (TFs), and chromatin states in discrete reprogramming stages and performed loss- and gain-of-function experiments. We found that OSK predominantly bind active somatic enhancers early in reprogramming and immediately initiate their inactivation genome-wide by inducing the redistribution of somatic TFs away from somatic enhancers to sites elsewhere engaged by OSK, recruiting Hdac1, and repressing the somatic TF Fra1. Pluripotency enhancer selection is a stepwise process that also begins early in reprogramming through collaborative binding of OSK at sites with high OSK-motif density. Most pluripotency enhancers are selected later in the process and require OS and other pluripotency TFs. Somatic and pluripotency TFs modulate reprogramming efficiency when overexpressed by altering OSK targeting, somatic-enhancer inactivation, and pluripotency enhancer selection. Together, our data indicate that collaborative interactions among OSK andwith stage-specific TFs direct both somatic-enhancer inactivation and pluripotency-enhancer selection to drive reprogramming.

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.

Multiple synaptotagmins are expressed in brain, but only synaptotagmins I and II have known functions in fast, synchronous Ca2+-triggered neurotransmitter release. Synaptotagmin III was proposed to regulate other aspects of synaptic vesicle exocytosis, particularly its slow component. Such a function predicts that synaptotagmin III should be an obligatory synaptic vesicle protein, as would also be anticipated from its high homology to synaptotagmins I and II. To test this hypothesis, we studied the distribution, developmental expression, and localization of synaptotagmin III and its closest homolog, synaptotagmin VI. We find that synaptotagmins III and VI are present in all brain regions in heterogeneous distributions and that their levels increase during development in parallel with synaptogenesis. Furthermore, we show by immunocytochemistry that synaptotagmin III is concentrated in synapses, as expected. Surprisingly, however, we observed that synaptotagmin III is highly enriched in synaptic plasma membranes but not in synaptic vesicles. Synaptotagmin VI was also found to be relatively excluded from synaptic vesicles. Our data suggest that synaptotagmins III and VI perform roles in neurons that are not linked to synaptic vesicle exocytosis but to other Ca2+-related nerve terminal events, indicating that the functions of synaptotagmins are more diverse than originally thought.

Thy-1, a glycosylphosphatidylinositol-linked integral membrane protein of the immunoglobulin superfamily, is a component of both large dense-core and small clear vesicles in PC12 cells. A majority of this protein, formerly recognized only on the plasma membrane of neurons, is localized to regulated secretory vesicles. Thy-1 is also present in synaptic vesicles in rat central nervous system. Experiments on permeabilized PC12 cells demonstrate that antibodies against Thy-1 inhibit the regulated release of neurotransmitter; this inhibition appears to be independent of any effect on the Ca2+ channel. These findings suggest Thy-1 is an integral component of many types of regulated secretory vesicles, and plays an important role in the regulated vesicular release of neurotransmitter at the synapse.

M-cadherin is a member of the multigene family of calcium-dependent intercellular adhesion molecules, the cadherins, which are involved in morphogenetic processes. Amino acid comparisons between M-cadherin and E-, N-, and P-cadherin suggested that M-cadherin diverged phylogenetically very early from these classical cadherins. It has been shown that M-cadherin is expressed in prenatal and adult skeletal muscle. In the cerebellum, M-cadherin is present in an adherens-type junction which differs in its molecular composition from the E-cadherin-mediated adherens-type junctions. These and other findings raised the question of whether M-cadherin and the classical cadherins share basic biochemical properties, notably the calcium-dependent resistance to proteolysis, mediation of calcium-dependent intercellular adhesion, and the capability to form M-cadherin complexes with the catenins. Here we show that M-cadherin is resistant to trypsin digestion in the presence of calcium ions but at lower trypsin concentrations than E-cadherin. When ectopically expressed in LMTK- cells, M-cadherin mediated calcium-dependent cell aggregation. Finally, M-cadherin was capable of forming two distinct cytoplasmic complexes in myogenic cells, either with alpha-catenin/beta-catenin or with alpha-catenin/plakoglobin, as E-and N-cadherin, for example, have previously been shown to form. The relative amount of these complexes changed during differentiation from C2C12 myoblasts to myotubes, although the molecular composition of each complex was unaffected during differentiation. These results demonstrate that M-cadherin shares important features with the classical cadherins despite its phylogenetic divergence.

Proteasomes are involved in the proteolytic generation of major histocompatibility complex (MHC) class I epitopes but their exact role has not been elucidated. We used highly purified murine 20S proteasomes for digestion of synthetic 22-mer and 41/44-mer ovalbumin partial sequences encompassing either an immunodominant or a marginally immunogenic epitope. At various times, digests were analyzed by pool sequencing and by semiquantitative electrospray ionization mass spectrometry. Most dual cleavage fragments derived from 22-mer peptides were 7-10 amino acids long, with octa- and nonamers predominating. Digestion of 41/44-mer peptides initially revealed major cleavage sites spaced by two size ranges, 8 or 9 amino acids and 14 or 15 amino acids, followed by further degradation of the latter as well as of larger single cleavage fragments. The final size distribution was slightly broader than that of fragments derived from 22-mer peptides. The majority of peptide bonds were cleaved, albeit with vastly different efficiencies. This resulted in multiple overlapping proteolytic fragments including a limited number of abundant peptides. The immunodominant epitope was generated abundantly whereas only small amounts of the marginally immunogenic epitope were detected. The frequency distributions of amino acids flanking proteasomal cleavage sites are correlated to that reported for corresponding positions of MHC class I binding peptides. The results suggest that proteasomal degradation products may include fragments with structural properties similar to MHC class I binding peptides. Proteasomes may thus be involved in the final stages of proteolytic epitope generation, often without the need for downstream proteolytic events.

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.

Major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL) recognize peptide epitopes of protein antigens in a hierarchical fashion. We investigated whether proteolytic cleavage, in particular by proteasomes, Is important in determining epitope hierarchy. Using highly purified 20S proteasomes, we find preferred cleavage sites directly adjacent to the N- and C-terminal ends of the immunodominant epitope of chicken ovalbumin, Ova257–264, while most of the subdominant epitope, Ova55–62, is destroyed by a major cleavage site located within this epitope. Moreover, we show that variations in amino acid sequences flanking these epitopes influence proteasomal cleavage patterns In parallel with the efficacy of their presentation. The results suggest that proteasomal cleavage within and adjacent to class I-restricted epitopes contributes to their level of presentation.

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.

Hematopoietic stem cells (HSC) are characterized with the capacity for self-renewal as well as multi-lineage differentiation, maintaining the immune system and blood cell formation throughout life. Although studies for the HSC biology have been in the forefront of the stem cell research field, many questions still remain with regard to the origin, development, and aging of HSC. Furthermore, needless to say, HSC are very useful for clinical medicine, particularly in the transplantation and/or regeneration therapy for hematological malignancies. Success of those therapies depends on how effectively HSC are purified and transplanted to the patients. In order to address those important issues in both basic and clinical science, information of cell surface molecules that selectively mark HSC is essential.

The cytoplasmic domain of classical cadherins is tightly associated with three proteins termed alpha-, beta- and gamma-catenin. These accessory proteins are of central importance for the adhesive properties of this class of cell adhesion molecules. In order to examine the molecular architecture of the cadherin-catenin complex in more detail we have expressed the catenins and the cytoplasmic domain of E-cadherin as fusion proteins in Escherichia coli, and analyzed the interaction of purified recombinant cadherin and catenins in combinatorial protein-protein interaction experiments. The cytoplasmic domain of E-cadherin cannot directly associate with alpha-catenin but interacts with high affinity with beta-catenin, whereas the binding of gamma-catenin (plakoglobin) to E-cadherin is less efficient. alpha- and beta-catenin assemble into a 1:1 heterodimeric complex. The analysis of various truncated beta-catenins revealed that an alpha-catenin binding site in beta-catenin is localized between amino acid positions 120 and 151. The central role of beta-catenin for the assembly of the heterotrimeric E-cadherin/alpha-catenin/beta-catenin complex in mixing experiments with all components was demonstrated. The reconstitution in vitro of the cadherin-catenin complex should allow the study of the interaction with signalling molecules and with the actin-based cytoskeleton.

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.

The right fit: Plasmid DNA molecules show chirality-dependent interaction with gold surfaces modified by L and D N-isobutyrylcysteine. Relaxed DNA molecules have a stronger interaction and adsorption on the L surface, while their counterparts on the D surface maintain a supercoiled conformation, indicating a weak interaction (see picture).

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.

Background: The endothelial receptors that control leukocyte transmigration in the postischemic liver are not identified. 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 and Results: The role of ESAM for leukocyte migration in the liver was analyzed using ESAM-deficient mice in a model of warm hepatic ischemia-reperfusion (90 min/30–360 min). 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. The postischemic neutrophil transmigration was significantly attenuated in ESAM−/− mice after 2 h of reperfusion, whereas it was completely restored after 6 h. 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 30 min 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.

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.

Endothelial cell–selective adhesion molecule (ESAM) is specifically expressed at endothelial tight junctions and on platelets. To test whether ESAM is involved in leukocyte extravasation, we have generated mice carrying a disrupted ESAM gene and analyzed them in three different inflammation models. We found that recruitment of lymphocytes into inflamed skin was unaffected by the gene disruption. However, the migration of neutrophils into chemically inflamed peritoneum was inhibited by 70% at 2 h after stimulation, recovering at later time points. Analyzing neutrophil extravasation directly by intravital microscopy in the cremaster muscle revealed that leukocyte extravasation was reduced (50%) in ESAM−/− mice without affecting leukocyte rolling and adhesion. Depletion of >98% of circulating platelets did not abolish the ESAM deficiency–related inhibitory effect on neutrophil extravasation, indicating that it is only ESAM at endothelial tight junctions that is relevant for the extravasation process. Knocking down ESAM expression in endothelial cells resulted in reduced levels of activated Rho, a GTPase implicated in the destabilization of tight junctions. Indeed, vascular permeability stimulated by vascular endothelial growth factor was reduced in ESAM−/− mice. Collectively, ESAM at endothelial tight junctions participates in the migration of neutrophils through the vessel wall, possibly by influencing endothelial cell contacts.

The coxsackievirus-adenovirus receptor (CAR) is a cell contact protein on various cell types with unknown physiological function. It belongs to a subfamily of the immunoglobulin-superfamily of which some members are junctional adhesion molecules on epithelial and/or endothelial cells. CAR is dominantly expressed in the hearts and brains of mice until the newborne phase after which it becomes mainly restricted to various epithelial cells. To understand more about the physiological function of CAR, we have generated CAR-deficient mice by gene targeting. We found that these mice die between E11.5 and E13.5 of embryonal development. Ultrastructural analysis of cardiomyocytes revealed that the density of myofibrils was reduced and that their orientation and bundling was disorganized. In addition, mitochondria were enlarged and glycogen storage strongly enriched. In line with these defects, we observed pericardial edema formation as a clear sign of insufficient heart function. Developmental abnormalities likely to be secondary effects of gene ablation were the persistent singular cardial atrio-ventricular canal and dilatations of larger blood vessels such as the cardinal veins. The secondary nature of these defects was supported by the fact that CAR was not expressed on vascular cells or on cells of the vascular wall. No obvious signs for alterations of the histological organization of the placenta were observed. We conclude that CAR is required for embryonal heart development, most likely due to its function during the organization of myofibrils in cardiomyocytes.