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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., >or=2-fold up- or down-regulated, P
Normal human tissues, in addition to some tumors, express multiple different CD44 isoforms.
At least 20 different isoforms of the human CD44 lymphocyte-homing receptor/hyaluronan receptor have been described to date that arise from the differential splicing of up to 10 alternative exons (termed v1-v10) encoding the membrane-proximal extracellular domain. Although numerous analyses at the mRNA level have indicated tissue-specific expression of CD44 variants, few analyses have been performed at the protein level because of limited availability of suitable monoclonal antibodies. Recently, however, exon-specific monoclonal antibodies have been generated using bacterial fusion proteins, and these have been reported to detect high levels of vCD44 containing the v6 exon on human tumors. Together with earlier evidence linking this particular exon with tumor metastasis in the rat, these latter experiments have led to the interpretation that v6 splice variants play a causative role in tumor dissemination. In this paper we describe the use of a new and comprehensive panel of CD44 exon-specific monoclonal antibodies generated against a recombinant CD44(v3-10)-immunoglobulin chimera to study vCD44 expression in a large number of normal and neoplastic tissues. We show that the expression of vCD44 varies greatly among different human tumors and that some express either very low levels of vCD44 or no CD44 at all. Furthermore, we demonstrate that expression is not limited to isoforms containing the v6 exon but includes variants carrying v3, v4/5, and v8/9. Additionally, normal epithelial tissues are shown to express considerable levels of these same vCD44 isoforms. Such results argue against a ubiquitous role for vCD44 isoforms in promoting tumor growth and metastasis.
The identification of a new alternative exon with highly restricted tissue expression in transcripts encoding the mouse Pgp-1 (CD44) homing receptor. Comparison of all 10 variable exons between mouse, human, and rat.
The human CD44 cell surface glycoprotein family which has been implicated in lymphocyte homing, tumor metastasis, and extracellular matrix attachment consists of a large number of related isoforms that derive from the differential splicing of a single CD44 gene transcript. Recent mapping of the CD44 locus in man indicated the presence of at least nine alternative exons within the region of the gene encoding the variable membrane proximal extracellular domain. Here we report the identification of a 10th alternative exon (termed V1) in the mouse, human, and rat CD44 genes. We demonstrate tissue-specific patterns of expression for transcripts containing exons V1-V10 in the mouse and a highly restricted usage of exon V1 in transcripts from mouse gastric tissue. Close sequence homology between exons V1-V10 from mouse rat and human points to a specific functional role rather than a purely structural role for the membrane proximal extracellular domain of the CD44 molecule.
FOXC2 haploinsufficient mice are a model for human autosomal dominant lymphedema-distichiasis syndrome.
Lymphedema-distichiasis (LD) (OMIM 153400) is a rare autosomal-dominant condition characterized by pubertal onset of lower limb lymphedema and an aberrant second row of eyelashes arising from the meibomian glands. In some patients cardiac, skeletal and other defects coexist. We previously identified inactivating, nonsense and frameshift mutations in the forkhead transcription factor FOXC2 in affected members of LD families. To further delineate the relationship of FOXC2 deficiency to the clinical (and lymphangiodysplastic) phenotype in this syndrome, we performed dynamic lymphatic imaging and immunohistochemical examination of lymphatic tissues in mice heterozygous (+/-) for a targeted disruption of Foxc2. Adult heterozygote mice characteristically exhibited a generalized lymphatic vessel and lymph node hyper plasia and rarely exhibited hindlimb swelling. Retrograde lymph flow through apparently incompetent interlymphangion valves into the mesenteric nodes, intestinal wall and liver was also observed. In addition, Foxc2 +/- mice uniformly displayed distichiasis. We conclude that Foxc2 haploinsufficient mice mimic closely the distinctive lymphatic and ocular phenotype of LD patients. Furthermore, the craniofacial, cardiovascular and skeletal abnormalities sometimes associated with LD have previously been shown to be fully penetrant in homozygous Foxc2 null mice. This Foxc2 mutant mouse thus provides an ideal model for exploring molecular mechanisms and physiologic events in mesenchymal differentiation associated with lymphatic growth and development and the clinical abnormalities seen in human LD syndrome.
Intratumoral lymphatics are essential for the metastatic spread and prognosis in squamous cell carcinomas of the head and neck region.
Head and neck squamous cell carcinomas (HNSCCs) frequently disseminate to regional lymph nodes. To investigate the possible mechanisms involved, we studied the expression of cancer cell adhesion molecules together with lymphatic vascular and blood vascular markers in a panel of 97 primary HNSCC tumors and correlated expression levels with conventional clinicopathological parameters and with long-term prognosis. In particular, we measured the density of intratumoral and peritumoral lymph vessels as assessed with the marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and the density of tumor CD44, a receptor up-regulated in many metastatic cancers. Intratumoral LYVE-1(+) lymphatic vessels were clearly associated with a higher risk for local relapse as well as with poor disease-specific prognosis (P = 0.02 and 0.0009, respectively). In contrast, a high density of peritumoral LYVE-1(+) vessels was a sign of favorable survival (P = 0.05). Strong primary tumor CD44 expression was associated with a poor prognosis, an increased risk of local recurrence (P = 0.03 and 0.02, respectively), and an increase in resistance to radiation therapy (P = 0.03). CD44 was the only factor with an independent prognostic value for the disease-specific overall survival (P = 0.04). Our results suggest that intratumoral lymphatics play a greater role than peritumoral lymphatics in nodal metastasis of HNSCC and that tumor CD44 levels can predict sensitivity to radiation therapy. These parameters may be useful predictive and prognostic tools in the clinical management of HNSCC.
Blocking development of a CD8+ T cell response by targeting lymphatic recruitment of APC.
Generating a protective immune response to viral infection is known to depend upon the priming and clonal expansion of virus-specific CD8(+) T cells by Ag-loaded dendritic cells (DC) within secondary lymphoid tissue. However, the actual initiation of the response involves critical upstream events that control the recruitment of mature Ag-charged DC from the periphery via afferent lymphatics, events that are still only partly understood. Recent evidence has revealed that transmigration of lymphatic endothelium by DC is regulated by the adhesion molecules ICAM-1 and VCAM-1 both in vitro and in vivo. These findings imply that lymphatic entry may be an important rate-limiting step in primary immunity and a possible target for immune intervention. In this study, we have explored such possibilities using an F(5) TCR-transgenic mouse model to assess the contribution of lymphatic cell adhesion molecules in the CD8(+) T cell response to influenza virus nucleoprotein (NP). We show for the first time that immunization with ICAM-1- and VCAM-1-blocking mAbs can impair the T cell response in lymph node-draining sites of dermally administered nucleoprotein vaccine (MVA.HIVA.NP) by targeting lymphatic uptake of Ag-loaded DC ahead of other cell adhesion molecule-dependent events. These results reveal lymphatic entry as an important step that may be rate limiting in the development of immunity and reconfirm its potential as a target for localized immunotherapy in inflammation and tissue rejection.
Characterizing extravascular fluid transport of macromolecules in the tumor interstitium by magnetic resonance imaging.
Noninvasive imaging techniques to image and characterize delivery and transport of macromolecules through the extracellular matrix (ECM) and supporting stroma of a tumor are necessary to develop treatments that alter the porosity and integrity of the ECM for improved delivery of therapeutic agents and to understand factors which influence and control delivery, movement, and clearance of macromolecules. In this study, a noninvasive imaging technique was developed to characterize the delivery as well as interstitial transport of a macromolecular agent, albumin-GdDTPA, in the MCF-7 human breast cancer model in vivo, using magnetic resonance imaging. The transport parameters derived included vascular volume, permeability surface area product, macromolecular fluid exudate volume, and drainage and pooling rates. Immunohistochemical staining for the lymphatic endothelial marker LYVE-1 was done to determine the contribution of lymphatics to the macromolecular drainage. Distinct pooling and draining regions were detected in the tumors using magnetic resonance imaging. A few lymphatic vessels positively stained for LYVE-1 were also detected although these were primarily collapsed and tenuous suggesting that lymphatic drainage played a minimal role, and that the bulk of drainage was due to convective transport through the ECM in this tumor model.
Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis.
Metastasis is a frequent and lethal complication of cancer. Vascular endothelial growth factor-C (VEGF-C) is a recently described lymphangiogenic factor. Increased expression of VEGF-C in primary tumours correlates with dissemination of tumour cells to regional lymph nodes. However, a direct role for VEGF-C in tumour lymphangiogenesis and subsequent metastasis has yet to be demonstrated. Here we report the establishment of transgenic mice in which VEGF-C expression, driven by the rat insulin promoter (Rip), is targeted to beta-cells of the endocrine pancreas. In contrast to wild-type mice, which lack peri-insular lymphatics, RipVEGF-C transgenics develop an extensive network of lymphatics around the islets of Langerhans. These mice were crossed with Rip1Tag2 mice, which develop pancreatic beta-cell tumours that are neither lymphangiogenic nor metastatic. Double-transgenic mice formed tumours surrounded by well developed lymphatics, which frequently contained tumour cell masses of beta-cell origin. These mice frequently developed pancreatic lymph node metastases. Our findings demonstrate that VEGF-C-induced lymphangiogenesis mediates tumour cell dissemination and the formation of lymph node metastases.
Thrombospondin-1 selectively inhibits early-stage carcinogenesis and angiogenesis but not tumor lymphangiogenesis and lymphatic metastasis in transgenic mice.
The roles played by the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in the early stages of multi-step carcinogenesis and in the control of hematogenous versus lymphatic metastasis are unknown. To investigate these issues we compared tumor development in normal mice and in transgenic mice with targeted overexpression of TSP-1 in the epidermis following a standard two-step chemical skin carcinogenesis regimen. Overexpression of TSP-1 resulted in delayed and reduced development of premalignant epithelial hyperplasias, but did not inhibit the malignant conversion to squamous cell carcinomas. TSP-1 overexpression also suppressed tumor angiogenesis and distant organ metastasis, but failed to inhibit tumor-associated lymphangiogenesis or lymphatic tumor spread to regional lymph nodes. Concomitant with these results, we found that the endothelial TSP-1 receptor CD36 was mostly absent from cutaneous lymphatic vessels. Our findings indicate the potential use of TSP-1 for the prevention of premalignant stages of tumorigenesis and are likely to have implications for the further development of anti-angiogenic cancer therapies.
Characterization of a novel, stage-specific, invariant surface protein in Trypanosoma brucei containing an internal, serine-rich, repetitive motif.
A new surface membrane protein, invariant surface glycoprotein termed ISG100, was identified in Trypanosoma brucei, using catalyzed surface, radioiodination of intact cells. This integral membrane glycoprotein was purified by a combination of detergent extraction, lectin-affinity, and ion-exchange chromatography followed by preparative SDS-polyacrylamide gel electrophoresis. The protein was expressed only in bloodstream forms of the parasite, was heavily N-glycosylated, and was present in different clonal variants of the same serodeme as well as in different serodemes. The gene for this protein was isolated by screening a cDNA expression library with antibodies against the purified protein followed by screening of a genomic library. The nucleotide sequence of the gene (4050 base pairs) predicted a highly reiterative polypeptide containing three distinct domains, a unique N-terminal domain of about 10 kDa containing three potential N-glycosylation sites, which was followed by a large internal domain consisting entirely of 72 consecutive copies of a serine-rich, 17-amino acid motif (approximately 113 kDa) and terminated with an apparent transmembrane spanning region of about 3.3 kDa. The internal repeat region of this gene (3672 base pairs) represents the largest reiterative coding sequence to be fully characterized in any species of trypanosome. There was no significant homology with other known proteins, and overall the predicted protein was extremely hydrophobic. Unlike the genes for other surface proteins, the gene encoding ISG100 was present as a single copy. Although present in the flagellar pocket, ISG100 was predominantly associated with components of the pathways for endo/exocytosis, such as intracellular vesicles located in the proximity of the pocket as well a large, electron-lucent perinuclear digestive vacuole.
Increased expression of CD44v6 and CD44v3 in ulcerative colitis but not colonic Crohn's disease.
Immune mechanisms, possibly involving cell-surface molecules such as CD44, have been invoked to explain the pathogenesis of inflammatory bowel disease. We used monoclonal antibodies against epitopes encoded within the variable region of CD44 to investigate CD44 isoform expression in colon, small intestine, and liver in patients with various intestinal disorders and in controls. Biopsy samples from patients with ulcerative colitis showed significantly increased epithelial expression of CD44 isoforms containing the v6 and v3 epitopes, detected with antibodies 2F10 and 3G5, respectively. CD44v6 was detected on colonic crypt epithelial cells in 23 of 25 ulcerative colitis samples compared with 3 of 18 colonic Crohn's disease samples (p = 3.0 x 10(-6); odds ratio 57.5 [95% CI 6.83-702]) and 3 of 52 controls (22 normal colon, 10 infective colitis, 2 radiation colitis, and 18 colonic Crohn's disease; p < 1 x 10(-8); odds ratio 199 [25.5-2294]). No significant expression of CD44v6, CD44v3, or CD44v8/9 was found in samples of normal proximal colon from 4 patients with distal ulcerative colitis, whereas samples from the affected area showed staining for CD44v6 and CD44v3. No expression of CD44 variants was found in 15 samples of normal small intestine, 11 small-bowel pouchitis, 8 coeliac disease, 3 small-bowel Crohn's disease, 6 normal liver, 6 primary biliary cirrhosis, or 9 primary sclerosing cholangitis. The high intensity of CD44v6 and v3 epitope expression on crypt epithelial cells in ulcerative colitis suggests that CD44 isoforms may have an important role in ulcerative colitis. Their detection could have diagnostic potential in differentiating ulcerative colitis from other forms of colonic inflammation including Crohn's disease.
Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium.
The lymphatic vessel endothelial receptor LYVE-1 is implicated in the uptake of hyaluronan (HA) and trafficking of leukocytes to draining lymph nodes. Yet LYVE-1 has only weak affinity for hyaluronan and depends on receptor clustering and higher order ligand organization for durable binding in lymphatic endothelium. An unusual feature of LYVE-1 not found in other HA receptors is the potential to form disulfide-linked homodimers. However, their influence on function has not been investigated. Here we show LYVE-1 homodimers are the predominant configuration in lymphatic endothelium in vitro and in vivo, and formation solely requires the unpaired cysteine residue Cys-201 within the membrane-proximal domain, yielding a 15-fold higher HA binding affinity and an ∼67-fold slower off-rate than the monomer. Moreover, we show non-dimerizing LYVE-1 mutants fail to bind HA even when expressed at high densities in lymphatic endothelial cells or artificially cross-linked with antibody. Consistent with these findings, small angle X-ray scattering (SAXS) indicates the Cys-201 interchain disulfide forms a hinge that maintains the homodimer in an "open scissors" conformation, likely allowing arrangement of the two HA binding domains for mutual engagement with ligand. Finally, we demonstrate the Cys-201 interchain disulfide is highly labile, and selective reduction with TCEP-HCl disrupts LYVE-1 homodimers, ablating HA binding. These findings reveal binding is dependent not just on clustering but also on the biochemical properties of LYVE-1 homodimers. They also mark LYVE-1 as the first Link protein superfamily member requiring covalent homodimerization for function and suggest the interchain disulfide acts as a redox switch in vivo.
Characterization of a novel alanine-rich protein located in surface microdomains in Trypanosoma brucei.
Heterologous expression in COS cells followed by orientation-specific polymerase chain reaction to select and amplify cDNAs encoding surface proteins in Trypanosoma brucei resulted in the isolation of a cDNA ( approximately 1.4 kilobase) which encodes an acidic, alanine-rich polypeptide that is expressed only in bloodstream forms of the parasite and has been termed bloodstream stage alanine-rich protein (BARP). Analysis of the amino acid sequence predicted the presence of a typical NH(2)-terminal leader sequence as well as a COOH-terminal hydrophobic extension with the potential to be replaced by a glycosylphosphatidylinositol anchor. A search of existing protein sequences revealed partial homology between BARP and the major surface antigen of procyclic forms of Trypanosoma congolense. BARP migrated as a complex, heterogeneous series of bands on Western blots with an apparent molecular mass ( approximately 50-70 kDa) significantly higher than predicted from the amino acid sequence ( approximately 26 kDa). Confocal microscopy demonstrated that BARP was present in small discrete spots that were distributed over the entire cellular surface. Detergent extraction experiments revealed that BARP was recovered in the detergent-insoluble, glycolipid-enriched fraction. These data suggested that BARP may be sequestered in lipid rafts.
Rapid plasma membrane-endosomal trafficking of the lymph node sinus and high endothelial venule scavenger receptor/homing receptor stabilin-1 (FEEL-1/CLEVER-1).
The sinusoidal endothelia of liver, spleen, and lymph node are major sites for uptake and recycling of waste macromolecules through promiscuous binding to a disparate family of scavenger receptors. Among the most complex is stabilin-1, a large multidomain protein containing tandem fasciclin domains, epidermal growth factor-like repeats, and a C-type lectin-like hyaluronan-binding Link module, which functions as an endocytic receptor for acetylated low density lipoprotein and advanced glycation end products. Intriguingly, stabilin-1 has also been reported to mediate both homing of leukocytes across lymph node high endothelial venules and adhesion of metastatic tumor cells to peritumoral lymphatic vessels. Currently, however, it is not clear how stabilin-1 mediates these distinct functions. To address the issue, we have investigated the tissue and subcellular localization of stabilin-1 in detail and assessed the functional status of its Link module. We show that stabilin-1 is almost entirely intracellular in lymph node high endothelial venules, lymphatic sinus endothelium, and cultured endothelial cells but that a finite population, detectable only by fluorescent antibody or fluorescein-labeled (Fl)-acetylated low density lipoprotein uptake, cycles rapidly between the plasma membrane and EEA-1+ve (early endosome antigen 1) early endosomes. In addition, we show using full-length stabilin-1 cDNA and a stabilin-1/CD44 chimera in HeLa cells that intracellular targeting is influenced by the transmembrane domain/cytoplasmic tail, which contains a putative dileucine (DXXLL) Golgi to endosomal sorting signal. Finally, we provide evidence that the stabilin-1 Link domain binds neither hyaluronan nor other glycosaminoglycans. These properties support a role for stabilin-1 as a rapidly recycling scavenger receptor and argue against a role in cell adhesion or lymphocyte homing.
Molecular characterization of lymphatic endothelial cells.
The lymphatic microvasculature is uniquely adapted for the continuous removal of interstitial fluid and proteins and is an important entry point for leukocytes and tumor cells. Specialized functions of lymphatics suggest differences in the molecular composition of the lymphatic and blood vascular endothelium. However, the extent to which the two cell types differ is still unclear, and few molecules that are truly specific to lymphatic endothelial cells have been identified to date. We have isolated primary lymphatic and blood microvascular endothelial cells from human skin by immunoselection with the lymphatic marker LYVE-1 and demonstrate that the two cell lineages express distinct sets of vascular markers and respond differently to growth factors and extracellular matrix. Comparative microarray analysis of gene-expression profiles revealed a number of unique molecular properties that distinguish lymphatic and blood vascular endothelium. The molecular profile of lymphatic endothelium seems to reflect characteristic functional and structural features of the lymphatic capillaries. Classification of the differentially expressed genes into functional groups revealed particularly high levels of genes implicated in protein sorting and trafficking, indicating a more active role of lymphatic endothelium in uptake and transport of molecules than previously anticipated. The identification of a large number of genes selectively expressed by lymphatic endothelium should facilitate the discovery of hitherto unknown lymphatic vessel markers and provide a basis for the analysis of the molecular mechanisms accounting for the characteristic functions of lymphatic capillaries.
Regulation of blood and lymphatic vascular separation by signaling proteins SLP-76 and Syk.
Lymphatic vessels develop from specialized endothelial cells in preexisting blood vessels, but the molecular signals that regulate this separation are unknown. Here we identify a failure to separate emerging lymphatic vessels from blood vessels in mice lacking the hematopoietic signaling protein SLP-76 or Syk. Blood-lymphatic connections lead to embryonic hemorrhage and arteriovenous shunting. Expression of slp-76 could not be detected in endothelial cells, and blood-filled lymphatics also arose in wild-type mice reconstituted with SLP-76-deficient bone marrow. These studies reveal a hematopoietic signaling pathway required for separation of the two major vascular networks in mammals.
Rapid Lymphatic Dissemination of Encapsulated Group A Streptococci via Lymphatic Vessel Endothelial Receptor-1 Interaction.
The host lymphatic network represents an important conduit for pathogen dissemination. Indeed, the lethal human pathogen group A streptococcus has a predilection to induce pathology in the lymphatic system and draining lymph nodes, however the underlying basis and subsequent consequences for disease outcome are currently unknown. Here we report that the hyaluronan capsule of group A streptococci is a crucial virulence determinant for lymphatic tropism in vivo, and further, we identify the lymphatic vessel endothelial receptor-1 as the critical host receptor for capsular hyaluronan in the lymphatic system. Interference with this interaction in vivo impeded bacterial dissemination to local draining lymph nodes and, in the case of a hyper-encapsulated M18 strain, redirected streptococcal entry into the blood circulation, suggesting a pivotal role in the manifestation of streptococcal infections. Our results reveal a novel function for bacterial capsular polysaccharide in directing lymphatic tropism, with potential implications for disease pathology.