A new multimodality technique accurately maps the primary lymphatic landing sites of the bladder

Pathoanatomic studies have failed to map accurately the primary lymphatic landing sites of the urinary bladder.

Differential requirement for ROCK in dendritic cell migration within lymphatic capillaries in steady-state and inflammation

Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing red-fluorescent vasculature and yellow-fluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steady-state and inflammation.

Clinical, molecular, and genetic correlates of lymphatic spread in clear cell renal cell carcinoma

While it is well known that clear cell renal cell carcinoma (ccRCC) that presents with lymphatic spread is associated with an extremely poor prognosis, its molecular and genetic biology is poorly understood.

Lymphatic clearance of the human skin in patients with acute deep vein thrombosis using a novel fluorescent technique

The purpose of this study was to investigate lymphatic clearance of the human skin in patients with acute deep thrombosis of the femoral vein. In 13 patients with deep vein thrombosis and no other cause for swelling of the limbs, lymphatic clearance of the skin at the foot was measured. Ten microliters of fluorescein isothiocyanatedextran 150,000 were injected intradermally and the fluorescent light intensity of the deposit measured 10 min and 24 hours after injection by window densitometry. In addition, intralymphatic pressure was measured by the servo-nulling system. The results were compared with a sex- and age-matched control group. Fluorescent light intensity decreased by 23.8 +/- 12.3 arbitrary units or by a factor of 1.8 +/- 0.5 in patients with DVT after 24 hours, which was significantly less than in healthy controls (33.7 +/- 8.9 arbitrary units or by factor 5.0 +/- 4.1, p < 0.013). Intralymphatic pressure was not different between the two groups. These results indicate that lymphatic clearance is significantly reduced in the acute phase of deep venous thrombosis.

The template of the primary lymphatic landing sites of the prostate should be revisited: results of a multimodality mapping study

OBJECTIVES: To map the primary prostatic lymphatic landing sites using a multimodality technique. METHODS: Thirty-four patients with organ-confined prostate cancer (cT1-cT2; cN0) underwent single-photon emission computed tomography fused with data from computed tomography (SPECT/CT) (n=33) or magnetic resonance imaging (SPECT/MRI) (n=1) 1h after ultrasound-guided intraprostatic injection of technecium (Tc-99m) nanocolloid. The presence of lymph nodes (LNs) containing Tc-99m was confirmed intraoperatively with a gamma probe. A backup extended pelvic lymphadenectomy (PLND) was performed to preclude missed primary lymphatic landing sites. The SPECT/CT/MRI data sets were used to generate a three-dimensional projection of each LN site. RESULTS: A total of 317 LNs (median, 10 per patient; range, 3-19) were detected by SPECT/CT/MRI, 314 of which were confirmed by gamma probe. With an "extended" PLND, two thirds of all primary prostatic lymphatic landing sites are resected compared with only one third with a "limited" PLND. CONCLUSIONS: The multimodality technique presented here enables precise mapping of the primary prostatic lymphatic landing sites. PLND for prostate cancer should include not only the external and obturator regions as well as the portions medial and lateral to the internal iliac vessels, but also the common iliac LNs at least up to the ureteric crossing, thus removing approximately 75% of all nodes potentially harbouring metastasis.

Distinct molecular composition of blood and lymphatic vascular endothelial cell junctions establishes specific functional barriers within the peripheral lymph node

Lymph nodes are strategically localized at the interfaces between the blood and lymphatic vascular system, delivering immune cells and antigens to the lymph node. As cellular junctions of endothelial cells actively regulate vascular permeability and cell traffic, we have investigated their molecular composition by performing an extensive immunofluorescence study for adherens and tight junction molecules, including vascular endothelium (VE)-cadherin, the vascular claudins 1, 3, 5 and 12, occludin, members of the junctional adhesion molecule family plus endothelial cell-selective adhesion molecule (ESAM)-1, platelet endothelial cell adhesion molecule-1, ZO-1 and ZO-2. We found that junctions of high endothelial venules (HEV), which serve as entry site for naive lymphocytes, are unique due to their lack of the endothelial cell-specific claudin-5. LYVE-1(+) sinus-lining endothelial cells form a diffusion barrier for soluble molecules that arrive at the afferent lymph and use claudin-5 and ESAM-1 to establish characteristic tight junctions. Analysis of the spatial relationship between the different vascular compartments revealed that HEV extend beyond the paracortex into the medullary sinuses, where they are protected from direct contact with the lymph by sinus-lining endothelial cells. The specific molecular architecture of cellular junctions present in blood and lymphatic vessel endothelium in peripheral lymph nodes establishes distinct barriers controlling the distribution of antigens and immune cells within this tissue.

PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature

The transmembrane ligand ephrinB2 and its cognate Eph receptor tyrosine kinases are important regulators of embryonic blood vascular morphogenesis. However, the molecular mechanisms required for ephrinB2 transduced cellular signaling in vivo have not been characterized. To address this question, we generated two sets of knock-in mice: ephrinB2DeltaV mice expressed ephrinB2 lacking the C-terminal PDZ interaction site, and ephrinB2(5F) mice expressed ephrinB2 in which the five conserved tyrosine residues were replaced by phenylalanine to disrupt phosphotyrosine-dependent signaling events. Our analysis revealed that the homozygous mutant mice survived the requirement of ephrinB2 in embryonic blood vascular remodeling. However, ephrinB2DeltaV/DeltaV mice exhibited major lymphatic defects, including a failure to remodel their primary lymphatic capillary plexus into a hierarchical vessel network, hyperplasia, and lack of luminal valve formation. Unexpectedly, ephrinB2(5F/5F) mice displayed only a mild lymphatic phenotype. Our studies define ephrinB2 as an essential regulator of lymphatic development and indicate that interactions with PDZ domain effectors are required to mediate its functions.

The chemokine receptors ACKR2 and CCR2 reciprocally regulate lymphatic vessel density

Macrophages regulate lymphatic vasculature development; however, the molecular mechanisms regulating their recruitment to developing, and adult, lymphatic vascular sites are not known. Here, we report that resting mice deficient for the inflammatory chemokine-scavenging receptor, ACKR2, display increased lymphatic vessel density in a range of tissues under resting and regenerating conditions. This appears not to alter dendritic cell migration to draining lymph nodes but is associated with enhanced fluid drainage from peripheral tissues and thus with a hypotensive phenotype. Examination of embryonic skin revealed that this lymphatic vessel density phenotype is developmentally established. Further studies indicated that macrophages and the inflammatory CC-chemokine CCL2, which is scavenged by ACKR2, are associated with this phenotype. Accordingly, mice deficient for the CCL2 signalling receptor, CCR2, displayed a reciprocal phenotype of reduced lymphatic vessel density. Further examination revealed that proximity of pro-lymphangiogenic macrophages to developing lymphatic vessel surfaces is increased in ACKR2-deficient mice and reduced in CCR2-deficient mice. Therefore, these receptors regulate vessel density by reciprocally modulating pro-lymphangiogenic macrophage recruitment, and proximity, to developing, resting and regenerating lymphatic vessels.

Blood and lymphatic vessel invasion in pT1 colorectal cancer: an international concordance study

AIM This study was performed to evaluate the concordance in pathological assessments of blood and lymphatic vessel invasion (BLI) in pT1 colorectal cancers and to assess the effect of diagnostic criterion on consistency in the assessment of BLI. METHODS Forty consecutive patients undergoing surgical resection of pT1 colorectal cancers were entered into this study. H&E-stained, D2-40-stained and elastica-stained slides from the tumours were examined by 18 pathologists from seven countries. The 40 cases were divided into two cohorts with 20 cases each. In cohort 1, pathologists diagnosed BLI using criteria familiar to them; all Japanese pathologists used a criterion of BLI from the Japanese Society for Cancer of the Colon and Rectum (JSCCR). In cohort 2, all pathologists used the JSCCR diagnostic criterion. RESULTS In cohort 1, diagnostic concordance was moderate in the US/Canadian and European pathologists. There were no differences in the consistency compared with results for Japanese pathologists, and no improvement in the diagnostic concordance was found for using the JSCCR criterion. However, in cohort 2, the JSCCR criterion decreased the consistency of BLI diagnosis in the US/Canadian and European pathologists. The level of decreased consistency in the assessment of BLI was different between the US/Canadian and European pathologists. CONCLUSIONS A uniform criterion strongly influences the diagnostic consistency of BLI but may not always improve the concordance. Further study is required to achieve an objective diagnosis of BLI in colorectal cancer. The varying effects of diagnostic criterion on the pathologists from Japan, the USA/Canada and Europe might reflect varied interpretations of the criterion. Internationally accepted criterion should be developed by participants from around the world.

FOXC2 and fluid shear stress stabilize postnatal lymphatic vasculature

Biomechanical forces, such as fluid shear stress, govern multiple aspects of endothelial cell biology. In blood vessels, disturbed flow is associated with vascular diseases, such as atherosclerosis, and promotes endothelial cell proliferation and apoptosis. Here, we identified an important role for disturbed flow in lymphatic vessels, in which it cooperates with the transcription factor FOXC2 to ensure lifelong stability of the lymphatic vasculature. In cultured lymphatic endothelial cells, FOXC2 inactivation conferred abnormal shear stress sensing, promoting junction disassembly and entry into the cell cycle. Loss of FOXC2-dependent quiescence was mediated by the Hippo pathway transcriptional coactivator TAZ and, ultimately, led to cell death. In murine models, inducible deletion of Foxc2 within the lymphatic vasculature led to cell-cell junction defects, regression of valves, and focal vascular lumen collapse, which triggered generalized lymphatic vascular dysfunction and lethality. Together, our work describes a fundamental mechanism by which FOXC2 and oscillatory shear stress maintain lymphatic endothelial cell quiescence through intercellular junction and cytoskeleton stabilization and provides an essential link between biomechanical forces and endothelial cell identity that is necessary for postnatal vessel homeostasis. As FOXC2 is mutated in lymphedema-distichiasis syndrome, our data also underscore the role of impaired mechanotransduction in the pathology of this hereditary human disease.

Attenuated portal hypertension in germ-free mice: Function of bacterial flora on the development of mesenteric lymphatic and blood vessels

Intestinal bacterial flora may induce splanchnic hemodynamic and histological alterations that are associated with portal hypertension (PH). We hypothesized that experimental PH would be attenuated in the complete absence of intestinal bacteria. We induced prehepatic PH by partial portal vein ligation (PPVL) in germ-free (GF) or mice colonized with altered Schaedler's flora (ASF). After 2 or 7 days, we performed hemodynamic measurements, including portal pressure (PP) and portosystemic shunts (PSS), and collected tissues for histomorphology, microbiology, and gene expression studies. Mice colonized with intestinal microbiota presented significantly higher PP levels after PPVL, compared to GF, mice. Presence of bacterial flora was also associated with significantly increased PSS and spleen weight. However, there were no hemodynamic differences between sham-operated mice in the presence or absence of intestinal flora. Bacterial translocation to the spleen was demonstrated 2 days, but not 7 days, after PPVL. Intestinal lymphatic and blood vessels were more abundant in colonized and in portal hypertensive mice, as compared to GF and sham-operated mice. Expression of the intestinal antimicrobial peptide, angiogenin-4, was suppressed in GF mice, but increased significantly after PPVL, whereas other angiogenic factors remained unchanged. Moreover, colonization of GF mice with ASF 2 days after PPVL led to a significant increase in intestinal blood vessels, compared to controls. The relative increase in PP after PPVL in ASF and specific pathogen-free mice was not significantly different. CONCLUSION In the complete absence of gut microbial flora PP is normal, but experimental PH is significantly attenuated. Intestinal mucosal lymphatic and blood vessels induced by bacterial colonization may contribute to development of PH.

Indocyanine Green Fluorescence Imaging in the Surgical Management of an Iatrogenic Lymphatic Fistula: Description of a Surgical Technique

We present a case of laparoscopic surgical management of an iatrogenic lymphorrhea using indocyanine green (ICG). A case of a patient who developed recurrent symptomatic lymphorrhea after laparoscopic radical hysterectomy and bilateral pelvic lymphadenectomy for an early stage cervical cancer is presented. Intraoperative bipedal interdigital subcutaneous injection of ICG exactly localized the disrupted lymphatic duct on fluorescence imaging performed with a near-infrared laparoscopic fluorescent optic device, thus allowing a successful surgical repair.

Insights into the biology of lymphatic metastasis

Recent data suggest that the generation of new lymphatic vessels (i.e. lymphangiogenesis) may be a rate-limiting step in the dissemination of tumor cells to regional lymph nodes. However, efforts to study the cellular and molecular interactions that take place between tumor cells and lymphatic endothelial cells have been limited due to a lack of lymphatic endothelial cell lines available for study. ^ I have used a microsurgical approach to establish conditionally immortalized lymphatic endothelial cell lines from the afferent mesenteric lymphatic vessels of mice. Characterization of lymphatic endothelial cells, and tumor-associated lymphatic vessels revealed high expression levels of VCAM-1, which is known to facilitate adhesion of some tumor cells to vascular endothelial cells. Further investigation revealed that murine melanoma cells selected for high expression of α4, a counter-receptor for VCAM-1, demonstrated enhanced adhesion to lymphatic endothelial cells in vitro, and increased tumorigenicity and lymphatic metastasis in vivo, despite similar lymphatic vessel numbers. ^ Next, I examined the effects of growth factors that regulate lymphangiogenesis, and report that several growth factors are capable of activating survival and proliferation pathways of lymphatic endothelial cells. The dual protein tyrosine kinase inhibitor AEE788 (EGFR and VEGFR-2) inhibited the activation of Akt and MAPK in lymphatic endothelial cells responding to multiple growth factors. Moreover, oral treatment of mice with AEE788 decreased lymphatic vessel density and production of lymphatic metastasis by human colon cancer cells growing in the cecum of nude mice. ^ In the last set of experiments, I investigated the surgical management of lymphatic metastasis using a novel model of sentinel lymphadenectomy in live mice bearing subcutaneous B16-BL6 melanoma. The data demonstrate that this procedure when combined with wide excision of the primary melanoma, significantly enhanced survival of syngeneic C57BL/6 mice. ^ Collectively, these results indicate that the production of lymphatic metastasis depends on lymphangiogenesis, tumor cell adhesion to lymphatic endothelial cells, and proliferation of tumor cells in lymph nodes. Thus, lymphatic metastasis is a multi-step, complex, and active process that depends upon multiple interactions between tumor cells and tumor associated lymphatic endothelial cells. ^

A physiologic function for p-glycoprotein (MDR-1) during the migration of dendritic cells from skin via afferent lymphatic vessels

P-glycoprotein (MDR-1) is a well-known transporter that mediates efflux of chemotherapeutic agents from the intracellular milieu and thereby contributes to drug resistance. MDR-1 also is expressed by nonmalignant cells, including leukocytes, but physiologic functions for MDR-1 are poorly defined. Using an initial screening assay that included >100 mAbs, we observed that neutralizing mAbs MRK16, UIC2, and 4E3 against MDR-1 specifically and potently blocked basal-to-apical transendothelial migration of mononuclear phagocytes, a process that may mimic their migration into lymphatic vessels. Antagonists of MDR-1 then were used in a model of authentic lymphatic clearance. In this model, antigen-presenting dendritic cells (DC) migrate out of explants of cultured human skin and into the culture medium via dermal lymphatic vessels. DC and T cells derived from skin expressed MDR-1 on their surfaces. Addition of anti-MDR-1 mAbs MRK16, UIC2, or the MDR-1 antagonist verapamil to skin explants at the onset of culture inhibited the appearance of DC, and accompanying T cells, in the culture medium by approximately 70%. Isotype-matched control mAbs against other DC molecules including CD18, CD31, and major histocompatibility complex I did not block. In the presence of MDR-1 antagonists, epidermal DC were retained in the epidermis, in contrast to control conditions. In summary, this work identifies a physiologic function for MDR-1 during the mobilization of DC and begins to elucidate how these critical antigen-presenting cells migrate from the periphery to lymph nodes to initiate T lymphocyte-mediated immunity.

Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development.

We have recently cloned the human fms-like tyrosine kinase 4 gene FLT4, whose protein product is related to two vascular endothelial growth factor receptors FLT1 and KDR/FLK1. Here the expression of FLT4 has been analyzed by in situ hybridization during mouse embryogenesis and in adult human tissues. The FLT4 mRNA signals first became detectable in the angioblasts of head mesenchyme, the cardinal vein, and extraembryonally in the allantois of 8.5-day postcoitus (p.c.) embryos. In 12.5-day p.c. embryos, the FLT4 signal decorated developing venous and presumptive lymphatic endothelia, but arterial endothelia were negative. During later stages of development, FLT4 mRNA became restricted to vascular plexuses devoid of red cells, representing developing lymphatic vessels. Only the lymphatic endothelia and some high endothelial venules expressed FLT4 mRNA in adult human tissues. Increased expression occurred in lymphatic sinuses in metastatic lymph nodes and in lymphangioma. Our results suggest that FLT4 is a marker for lymphatic vessels and some high endothelial venules in human adult tissues. They also support the theory on the venous origin of lymphatic vessels.