Lymphatic Patterning Research Articles

Lymphatic endothelial cell sphingosine kinase activity is required for lymphocyte egress and lymphatic patterning

Lymphocyte egress from lymph nodes (LNs) is dependent on sphingosine-1-phosphate (S1P), but the cellular source of this S1P is not defined. We generated mice that expressed Cre from the lymphatic vessel endothelial hyaluronan receptor 1 (Lyve-1) locus and that showed efficient recombination of loxP-flanked genes in lymphatic endothelium. We report that mice with Lyve-1 CRE-mediated ablation of sphingosine kinase (Sphk) 1 and lacking Sphk2 have a loss of S1P in lymph while maintaining normal plasma S1P. In Lyve-1 Cre+ Sphk-deficient mice, lymphocyte egress from LNs and Peyer's patches is blocked. Treatment with pertussis toxin to overcome Gαi-mediated retention signals restores lymphocyte egress. Furthermore, in the absence of lymphatic Sphks, the initial lymphatic vessels in nonlymphoid tissues show an irregular morphology and a less organized vascular endothelial cadherin distribution at cell–cell junctions. Our data provide evidence that lymphatic endothelial cells are an in vivo source of S1P required for lymphocyte egress from LNs and Peyer's patches, and suggest a role for S1P in lymphatic vessel maturation.

NFATc1 regulates lymphatic endothelial development

NFATc1 transcription factor is critical for lineage selection in T-cell differentiation, cardiac valve morphogenesis and osteoclastogenesis. We identified a role for calcineurin–NFAT signaling in lymphatic development and patterning. NFATc1 was colocalized with lymphatic markers Prox-1, VEGFR-3 and podoplanin on cardinal vein as lymphatic endothelial cells (LEC) are specified and as they segregate into lymph sacs and mature lymphatics. In NFATc1 null mice, Prox-1, VEGFR-3 and podoplanin positive endothelial cells sprouted from the cardinal vein at E11.5, but poorly coalesced into lymph sacs. NFAT activation requires the phosphatase calcineurin. Embryos treated in utero with the calcineurin inhibitor cyclosporine-A showed cytoplasmic NFATc1, diminished podoplanin and FGFR-3 expression by the lymphatics and irregular patterning of the LEC sprouts coming off the jugular lymph sac, which suggests a role for calcineurin–NFAT signaling in lymphatic patterning. In a murine model of injury-induced lymphangiogenesis, NFATc1 was expressed on the neolymphatics induced by lung-specific overexpression of VEGF-A. Mice lacking the calcineurin Aβ regulatory subunit, with diminished nuclear NFAT, failed to respond to VEGF-A with increased lymphangiogenesis. In vitro, endogenous and VEGF-A-induced VEGFR-3 and podoplanin expression by human microvascular endothelial cells was reduced by siRNA to NFATc1, to levels comparable to reductions seen with siRNA to Prox-1. In reporter assays, NFATc1 activated lymphatic specific gene promoters. These results demonstrate the role of calcineurin–NFAT pathway in lymphangiogenesis and suggest that NFATc1 is the principle NFAT involved.

Functional Difference of Two Tie2 Ligands, Angiopoietin-1 and -2, in the Regulation of the Adult Bone Marrow Hematopoietic Stem Cells

Majority of the adult HSCs is in quiescent state in cell cycle, and it is likely that maintenance of the HSC quiescence and slow cell-cycle progression are critical for sustaining the self-renewing HSC pool. Indeed, sub-populations of long-term HSC (LT-HSC) are in the quiescent state and have robust reconstitution activity of hematopoiesis. Therefore, it is suggested that quiescence of HSCs is directly linked to the ability for the efficient reconstitution. We previously reported that the side-population (SP) in Lineage− Sca-1+Kit+ (LSK) cells resided in the BM niche is the quiescent HSC population, and is enriched in the Tie2 receptor-positive fraction in LSK. So far, four types of ligands, angiopoietin (Ang)-1~4, have been identified as the ligands of Tie2. We found that Ang-1/Tie2 signaling induces the quiescence or slow cell cycling of HSCs and contributes to the maintenance of long-term repopulating ability of LT-HSCs (Arai et al. 2004). Ang-2 is dispensable for embryonic angiogenesis but is specifically required for postnatal vascular remodeling and lymphatic patterning. In the hematopoietic system, however, the role of Ang-2 in the regulation of LT-HSCs is still unclear. Here, we investigated the functional difference between Ang-1 and Ang-2 in the maintenance of LT-HSCs. First, we examined the expression of CDK inhibitors, cell adhesion molecules, cytokine receptors, transcription factors, and anti-apoptotic factors induced by the Ang-1 and/or Ang2. LSK-SP cells were cultured with stem cell factor (SCF) in the presence or absence of angiopoietins (Ang-1 and/or Ang-2), and gene expressions were analyzed by high-throughput real-time PCR assays using a novel nanofluidic platform (BioMark™ Dynamic Array and Digital Array, Fluidigm). We found that Ang-1 upregulated the expression of p57, p18, α4-integrin, β1-integrin, N-cadherin, Tie2, Hoxb4, Bmi-1 and Bax, compared to control. Although Ang-2 also upregulated p57 and Tie2, its effect was smaller than that of Ang-1. In addition, Ang-1-induced upregulations of p57 and p18 were suppressed by the simultaneous addition of Ang-2. Next we examined the effects of Ang-1 and Ang-2 on the long-term engraftment capacity of LSK-SP cells. LSK-SP cells (Ly5.1+) were cultured in the serum-free medium containing SCF and thrombopoietin (THPO) with or without angiopoietins (Ang-1 and/or Ang-2). After 7 days of culture, cells were transplanted into the recipient mice (Ly5.2+). Percentage of Ly5.1+ cells in the mice transplanted with the HSCs cultured with SCF and THPO was 9.8 ± 4.0 % after 3 months of BM transplantation (BMT). Consistent with our previous report, the LSK-SP cells cultured with Ang-1 efficiently maintained long-term repopulation (LTR), and the percentage of Ly5.1+ cells in the recipient's peripheral blood was 40.8 ± 7.7 % after 3 months of BMT. Although Ang-2 also maintained LTR activity of HSCs (14.5 ± 4.7 %), the frequency of donor-derived cells was significantly lower than the case of Ang-1. Furthermore, the effect of Ang-1 was dramatically interfered by the addition of Ang-2 (10.0 ± 4.5 %). These observations suggest that in the BM niche, especially in the endosteal niche, Ang-1 might be the dominant ligand for Tie2 in LT-HSCs and contribute to the maintenance of the stem cell activities. In addition, we isolated osteoblasts (OB) and mesenchymal progenitor cells (MPCs), the major osteoblastic niche components, from endosteum by collagenase treatment of bone fragments and found that osteoblastic niche cells expressed Ang-1 but not Ang-2, which is consistent with the results of qPCR and LTR-assay. We hypothesized that Ang-2 is involved in the release of HSCs from the niche for the mobilization of HSCs. Collectively, Ang-1 plays a role as the key factor for LTR-activity maintenance of LT-HSCs, and Ang-2 has an antagonistic effect against Ang-1/Tie2 signaling.

PR07 A SYSTEM FOR QUANTIFYING LYMPHATIC VESSEL ABNORMALITIES IN LYMPHEDEMA MODELS, AND LOCO‐SYSTEMIC EFFECTS OF PRO‐LYMPHATIC TUMOR GROWTH FACTORS

Purpose Lymphatic vessels are critical in interstitial fluid balance, and congenital or acquired derangements lead to accumulation of lymph fluid (lymphedema). Further, when induced by pro‐lymphatic growth factors in tumors, they facilitate lymph node metastasis(1). While histological vessel density is counted on tissue sections, there is no method for quantifying lymphatic morphology and patterning in 3‐D; key elements in fluid dynamics and cellular interactions that underlie lymphatic function and diseases resulting from alterations to them. We have developed a method to quantify important biological and functional parameters in lymphatics, to help distinguish subtle phenotypic differences and patterning defects.Methods Apical images were taken of whole‐mounted mouse ears that were immunofluorescently stained for the lymphatic marker LYVE‐1. Key lymphatic parameters were quantified using a computer program we developed.Results Mice with gross lymphedema were found to have significant derangements of many of the key morphological and patterning characteristics assessed. Subtle yet statistically significant lymphatic abnormalities were also identified in less obviously lymphedematous animals. We were also able to assess loco‐systemic effects of the pro‐lymphatic growth factors.Conclusions Increasing emphasis on lymphatics in disease demands quantification methods that assess key biological and functional parameters. Our method allows comparison and identification of abnormal lymphatic phenotypes and allows assessment of the effects induced by pro‐lymphatic growth factors.

Up-Regulation of the Lymphatic Marker Podoplanin, a Mucin-Type Transmembrane Glycoprotein, in Human Squamous Cell Carcinomas and Germ Cell Tumors

The mucin-type glycoprotein podoplanin is specifically expressed by lymphatic but not blood vascular endothelial cells in culture and in tumor-associated lymphangiogenesis, and podoplanin deficiency results in congenital lymphedema and impaired lymphatic vascular patterning. However, research into the biological importance of podoplanin has been hampered by the lack of a generally available antibody against the human protein, and its expression in normal tissues and in human malignancies has remained unclear. We generated a human podoplanin-Fc fusion protein and found that the commercially available mouse monoclonal antibody D2-40 specifically recognized human podoplanin, as assessed by enzyme-linked immunosorbent assay and Western blot analyses. We found that, in addition to lymphatic endothelium, podoplanin was also expressed by peritoneal mesothelial cells, osteocytes, glandular myoepithelial cells, ependymal cells, and by stromal reticular cells and follicular dendritic cells of lymphoid organs. These findings were confirmed in normal mouse tissues with anti-podoplanin antibody 8.1.1. Podoplanin was also strongly expressed by granulosa cells in normal ovarian follicles, and by ovarian dysgerminomas and granulosa cell tumors. Although podoplanin was primarily absent from normal human epidermis, its expression was strongly induced in 22 of 28 squamous cell carcinomas studied. These findings suggest a potential role of podoplanin in tumor progression, and they also identify the first commercially available antibody for the specific staining of a defined lymphatic marker in archival human tissue sections, thereby enabling more widespread studies of tumor lymphangiogenesis in human cancers.

Angiopoietin-2 Is Required for Postnatal Angiogenesis and Lymphatic Patterning, and Only the Latter Role Is Rescued by Angiopoietin-1

VEGF and Angiopoietin-1 requisitely collaborate during blood vessel development. While Angiopoietin-1 obligately activates its Tie2 receptor, Angiopoietin-2 can activate Tie2 on some cells, while it blocks Tie2 activation on others. Our analysis of mice lacking Angiopoietin-2 reveals that Angiopoietin-2 is dispensable for embryonic vascular development but is requisite for subsequent angiogenic remodeling. Unexpectedly, mice lacking Angiopoietin-2 also exhibit major lymphatic vessel defects. Genetic rescue with Angiopoietin-1 corrects the lymphatic, but not the angiogenesis, defects, suggesting that Angiopoietin-2 acts as a Tie2 agonist in the former setting, but as an antagonist in the latter setting. Our studies define a vascular growth factor whose primary role is in postnatal angiogenic remodeling and also demonstrate that members of the VEGF and Angiopoietin families collaborate during development of the lymphatic vasculature.

Mesenchymal expression of vascular endothelial growth factors D and A defines vascular patterning in developing lung

The lung has specific vascular patterning requirements for effective gas exchange at birth, including alignment of airways and blood vessels and lymphatic vessels. Vascular endothelial growth factors (VEGF) are potent effectors of vascular development. We examined the temporal and spatial expression of VEGF-D and specific VEGF-A isoforms at each stage of lung development. VEGF-D, expressed only by cadherin-11-positive cells of the mesenchyme, is first detected at embryonic day (E) 13.5, a period of active vasculogenesis. VEGFR-3, its cognate receptor, is detected earlier on days E11.5 to E14.5, in both blood vessels and lymphatic vessels and later, on day E17.5, in only lymphatic vessels. VEGF-A is expressed in the mesenchyme throughout lung development and also by the epithelium midway through organogenesis. Before E14, the predominant forms of VEGF-A are the soluble isoforms, VEGF-A120 and 164. Not until E14.5 do epithelial cells at the tips of expanding airways express VEGF-A, including VEGF-A188, an isoform with high affinity for extracellular matrix. Our results demonstrate unique temporal and spatial expression of VEGF-D and specific VEGF-A isoforms during lung development and suggest these related factors have distinct functions in vascular and lymphatic patterning of the lung.