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Meningeal lymphatics are conduits for cerebrospinal fluid drainage to lymphatics and lymph nodes in the neck. In this issue of JEM, Boisserand et al. (https://doi.org/10.1084/jem.20221983) provide evidence that expansion of meningeal lymphatics protects against ischemic stroke.

Cerebrospinal fluid (CSF) in the subarachnoid space around the brain has long been known to drain through the lymphatics to cervical lymph nodes1-17, but the connections and regulation have been challenging to identify. Here, using fluorescent CSF tracers in Prox1-GFP lymphatic reporter mice18, we found that the nasopharyngeal lymphatic plexus is a major hub for CSF outflow to deep cervical lymph nodes. This plexus had unusual valves and short lymphangions but no smooth-muscle coverage, whereas downstream deep cervical lymphatics had typical semilunar valves, long lymphangions and smooth muscle coverage that transported CSF to the deep cervical lymph nodes. α-Adrenergic and nitric oxide signalling in the smooth muscle cells regulated CSF drainage through the transport properties of deep cervical lymphatics. During ageing, the nasopharyngeal lymphatic plexus atrophied, but deep cervical lymphatics were not similarly altered, and CSF outflow could still be increased by adrenergic or nitric oxide signalling. Single-cell analysis of gene expression in lymphatic endothelial cells of the nasopharyngeal plexus of aged mice revealed increased type I interferon signalling and other inflammatory cytokines. The importance of evidence for the nasopharyngeal lymphatic plexus functioning as a CSF outflow hub is highlighted by its regression during ageing. Yet, the ageing-resistant pharmacological activation of deep cervical lymphatic transport towards lymph nodes can still increase CSF outflow, offering an approach for augmenting CSF clearance in age-related neurological conditions in which greater efflux would be beneficial.

Leptomeninges, consisting of the pia mater and arachnoid, form a connective tissue investment and barrier enclosure of the brain. The exact nature of leptomeningeal cells has long been debated. In this study, we identify five molecularly distinct fibroblast-like transcriptomes in cerebral leptomeninges; link them to anatomically distinct cell types of the pia, inner arachnoid, outer arachnoid barrier, and dural border layer; and contrast them to a sixth fibroblast-like transcriptome present in the choroid plexus and median eminence. Newly identified transcriptional markers enabled molecular characterization of cell types responsible for adherence of arachnoid layers to one another and for the arachnoid barrier. These markers also proved useful in identifying the molecular features of leptomeningeal development, injury, and repair that were preserved or changed after traumatic brain injury. Together, the findings highlight the value of identifying fibroblast transcriptional subsets and their cellular locations toward advancing the understanding of leptomeningeal physiology and pathology.

BackgroundMutations in PIEZO1 (Piezo type mechanosensitive ion channel component 1) cause human lymphatic malformations. We have previously uncovered an ORAI1 (ORAI calcium release-activated calcium modulator 1)-mediated mechanotransduction pathway that triggers lymphatic sprouting through Notch downregulation in response to fluid flow. However, the identity of its upstream mechanosensor remains unknown. This study aimed to identify and characterize the molecular sensor that translates the flow-mediated external signal to the Orai1-regulated lymphatic expansion.MethodsVarious mutant mouse models, cellular, biochemical, and molecular biology tools, and a mouse tail lymphedema model were employed to elucidate the role of Piezo1 in flow-induced lymphatic growth and regeneration.ResultsPiezo1 was found to be abundantly expressed in lymphatic endothelial cells. Piezo1 knockdown in cultured lymphatic endothelial cells inhibited the laminar flow-induced calcium influx and abrogated the flow-mediated regulation of the Orai1 downstream genes, such as KLF2 (Krüppel-like factor 2), DTX1 (Deltex E3 ubiquitin ligase 1), DTX3L (Deltex E3 ubiquitin ligase 3L,) and NOTCH1 (Notch receptor 1), which are involved in lymphatic sprouting. Conversely, stimulation of Piezo1 activated the Orai1-regulated mechanotransduction in the absence of fluid flow. Piezo1-mediated mechanotransduction was significantly blocked by Orai1 inhibition, establishing the epistatic relationship between Piezo1 and Orai1. Lymphatic-specific conditional Piezo1 knockout largely phenocopied sprouting defects shown in Orai1- or Klf2- knockout lymphatics during embryo development. Postnatal deletion of Piezo1 induced lymphatic regression in adults. Ectopic Dtx3L expression rescued the lymphatic defects caused by Piezo1 knockout, affirming that the Piezo1 promotes lymphatic sprouting through Notch downregulation. Consistently, transgenic Piezo1 expression or pharmacological Piezo1 activation enhanced lymphatic sprouting. Finally, we assessed a potential therapeutic value of Piezo1 activation in lymphatic regeneration and found that a Piezo1 agonist, Yoda1, effectively suppressed postsurgical lymphedema development.ConclusionsPiezo1 is an upstream mechanosensor for the lymphatic mechanotransduction pathway and regulates lymphatic growth in response to external physical stimuli. Piezo1 activation presents a novel therapeutic opportunity for preventing postsurgical lymphedema. The Piezo1-regulated lymphangiogenesis mechanism offers a molecular basis for Piezo1-associated lymphatic malformation in humans.

Button-like junctions are discontinuous contacts at the border of oak-leaf-shaped endothelial cells of initial lymphatic vessels. These junctions are distinctively different from continuous zipper-like junctions that create the endothelial barrier in collecting lymphatics and blood vessels. Button junctions are point contacts, spaced about 3 µm apart, that border valve-like openings where fluid and immune cells enter lymphatics. In intestinal villi, openings between button junctions in lacteals also serve as entry routes for chylomicrons. Like zipper junctions that join endothelial cells, buttons consist of adherens junction proteins (VE-cadherin) and tight junction proteins (claudin-5, occludin, and others). Buttons in lymphatics form from zipper junctions during embryonic development, can convert into zippers in disease or after experimental genetic or pharmacological manipulation, and can revert back to buttons with treatment. Multiple signaling pathways and local microenvironmental factors have been found to contribute to button junction plasticity and could serve as therapeutic targets in pathological conditions ranging from pulmonary edema to obesity.

Changes in blood vessels and lymphatics in health and disease are easier to understand and interpret when studied microscopically in three dimensions. The mouse trachea is a simple, yet powerful, and versatile model system in which to achieve this. We describe practical immunohistochemical methods for fluorescence and confocal microscopy of wholemounts of the mouse trachea to achieve this purpose in which the entire vasculature can be visualized from the organ level to the cellular and subcellular level. Blood vessels and lymphatics have highly stereotyped vascular architectures that repeat in arcades between the tracheal cartilages. Arterioles, capillaries, and venules can be easily identified for the blood vessels, while the lymphatics consist of initial lymphatics and collecting lymphatics. Even small abnormalities in either blood vessels or lymphatics can be noticed and evaluated in three dimensions. We and others have used the mouse trachea for examining in situ angiogenesis and lymphangiogenesis, vascular development and regression, vessel patency, differences in transgenic mice, and pathological changes, such as increased vascular permeability induced by inflammatory mediators.

Oncolytic vaccinia viruses have promising efficacy and safety profiles in cancer therapy. Although antitumor activity can be increased by manipulating viral genes, the relative efficacy of individual modifications has been difficult to assess without side-by-side comparisons. This study sought to compare the initial antitumor activity after intravenous administration of five vaccinia virus variants of the same Western Reserve backbone and thymidine kinase gene deletion in RIP-Tag2 transgenic mice with spontaneous pancreatic neuroendocrine tumors. Tumors had focal regions of infection at 5 days after all viruses. Natural killer (NK) cells were restricted to these sites of infection, but CD8+ T cells and tumor cell apoptosis were widespread and varied among the viruses. Antitumor activity of virus VV-A34, bearing amino acid substitution A34K151E to increase viral spreading, and virus VV-IL2v, expressing a mouse IL2 variant (mIL2v) with attenuated IL2 receptor alpha subunit binding, was similar to control virus VV-GFP. However, antitumor activity was significantly greater after virus VV-A34/IL2v, which expressed mIL2v together with A34K151E mutation and viral B18R gene deletion, and virus VV-GMCSF that expressed mouse GM-CSF. Both viruses greatly increased expression of CD8 antigens Cd8a/Cd8b1 and cytotoxicity genes granzyme A, granzyme B, Fas ligand, and perforin-1 in tumors. VV-A34/IL2v led to higher serum IL2 and greater tumor expression of death receptor ligand TRAIL, but VV-GMCSF led to higher serum GM-CSF, greater expression of leukocyte chemokines and adhesion molecules, and more neutrophil recruitment. Together, the results show that antitumor activity is similarly increased by viral expression of GM-CSF or IL2v combined with additional genetic modifications.

Leakage from blood vessels into tissues is governed by mechanisms that control endothelial barrier function to maintain homeostasis. Dysregulated endothelial permeability contributes to many conditions and can influence disease morbidity and treatment. Diverse approaches used to study endothelial permeability have yielded a wealth of valuable insights. Yet, ongoing questions, technical challenges, and unresolved controversies relating to the mechanisms and relative contributions of barrier regulation, transendothelial sieving, and transport of fluid, solutes, and particulates complicate interpretations in the context of vascular physiology and pathophysiology. Here, we describe recent in vivo findings and other advances in understanding endothelial barrier function with the goal of identifying and reconciling controversies over cellular and molecular processes that regulate the vascular barrier in health and disease.

Despite many reports about pulmonary blood vessels in lung fibrosis, the contribution of lymphatics to fibrosis is unknown. We examined the mechanism and consequences of lymphatic remodeling in mice with lung fibrosis after bleomycin injury or telomere dysfunction. Widespread lymphangiogenesis was observed after bleomycin treatment and in fibrotic lungs of prospero homeobox 1-enhanced green fluorescent protein (Prox1-EGFP) transgenic mice with telomere dysfunction. In loss-of-function studies, blocking antibodies revealed that lymphangiogenesis 14 days after bleomycin treatment was dependent on vascular endothelial growth factor (Vegf) receptor 3 signaling, but not on Vegf receptor 2. Vegfc gene and protein expression increased specifically. Extensive extravasated plasma, platelets, and macrophages at sites of lymphatic growth were potential sources of Vegfc. Lymphangiogenesis peaked at 14 to 28 days after bleomycin challenge, was accompanied by doubling of chemokine (C-C motif) ligand 21 in lung lymphatics and tertiary lymphoid organ formation, and then decreased as lung injury resolved by 56 days. In gain-of-function studies, expansion of the lung lymphatic network by transgenic overexpression of Vegfc in club cell secretory protein (CCSP)/VEGF-C mice reduced macrophage accumulation and fibrosis and accelerated recovery after bleomycin treatment. These findings suggest that lymphatics have an overall protective effect in lung injury and fibrosis and fit with a mechanism whereby lung lymphatic network expansion reduces lymph stasis and increases clearance of fluid and cells, including profibrotic macrophages.

To have the desired therapeutic effect, nanomedicines and macromolecular medications must move from the site of injection to the site of action, without having adverse effects. Transvascular transport is a critical step of this navigation, as exemplified by the Enhanced Permeability and Retention (EPR) effect in solid tumors, not found in normal organs. Numerous studies have concluded that passive, diffusion- and convection-based transport predominates over active, cellular mechanisms in this effect. However, recent work using a new approach reevaluated this principle by comparing tumors with or without fixation and concluded the opposite. Here, we address the controversy generated by this new approach by reporting evidence from experimental investigations and computer simulations that separate the contributions of active and passive transport. Our findings indicate that tissue fixation reduces passive transport as well as active transport, indicating the need for new methods to distinguish the relative contributions of passive and active transport. [ABSTRACT FROM AUTHOR]

Zippering of cellular junctions in intestinal lacteals prevents fat uptake

Tumor lymphangiogenesis is accompanied by a higher incidence of sentinel lymph node metastasis and shorter overall survival in several types of cancer. We asked whether tumor lymphangiogenesis might also occur in distant organs with established metastases and whether it might promote further metastatic spread of those metastases to other organs. Using mouse metastasis models, we found that lymphangiogenesis occurred in distant lung metastases and that some metastatic tumor cells were located in lymphatic vessels and draining lymph nodes. In metastasis-bearing lungs of melanoma patients, a higher lymphatic density within and around metastases and lymphatic invasion correlated with poor outcome. Using a transgenic mouse model with inducible expression of vascular endothelial growth factor C (VEGF-C) in the lung, we found greater growth of lung metastases, with more abundant dissemination to other organs. Our findings reveal unexpected contributions of lymphatics in distant organs to the promotion of growth of metastases and their further spread to other organs, with potential clinical implications for adjuvant therapies in patients with metastatic cancer.

Oncolytic viruses pose many questions in their use in cancer therapy. In this study, we assessed the potential of mpJX-594 (mouse-prototype JX-594), a replication-competent vaccinia virus administered by intravenous injection, to target the tumor vasculature, produce immune activation and tumor cell killing more widespread than the infection, and suppress invasion and metastasis. These actions were examined in RIP-Tag2 transgenic mice with pancreatic neuroendocrine tumors that developed spontaneously and progressed as in humans. mpJX-594 initially infected tumor vascular endothelial cells, leading to vascular pruning and prolonged leakage in tumors but not in normal organs; parallel effects were observed in U87 gliomas. Viral infection spread to tumor cells, where tumor cell killing was much more widespread than the infection. Widespread tumor cell killing at 5 days was prevented by depletion of CD8+ T lymphocytes and did not require GM-CSF, as mpJX-594 variants that expressed human, mouse, or no GM-CSF produced equivalent amounts of killing. The antivascular, antitumor, and antimetastatic effects of mpJX-594 were amplified by concurrent or sequential administration of sunitinib, a multitargeted receptor tyrosine kinase inhibitor. These effects were not mimicked by selective inhibition of VEGFR2 despite equivalent vascular pruning, but were accompanied by suppression of regulatory T cells and greater influx of activated CD8+ T cells. Together, our results showed that mpJX-594 targets tumor blood vessels, spreads secondarily to tumor cells, and produces widespread CD8+ T-cell-dependent tumor cell killing in primary tumors and metastases, and that these effects can be amplified by coadministration of sunitinib.Significance: These findings reveal multiple unrecognized features of the antitumor properties of oncolytic vaccinia viruses, all of which can be amplified by the multitargeted kinase inhibitor sunitinib. Cancer Res; 78(4); 922-37. ©2017 AACR.

Lymphatic malformations and other conditions where lymphatic function is disturbed in the respiratory tract present diagnostic and therapeutic challenges. Advances in lymphatic development, growth regulation, function, and imaging have increased the understanding of lymphatics, but the airways and lungs have not received as much attentions as many other organs. The lung presents challenges for studies of lymphatics because of the complex, densely packed three-dimensional architecture of the airways and vasculature, and because it cannot readily be examined in its entirety. To address this problem, we developed methods for immunohistochemical examination of the lymphatics in mouse lungs, based on approaches we devised for lymphatic vessels and blood vessels in whole mounts of the mouse trachea. This report provides a practical guide for visualizing by fluorescence and confocal microscopy the lymphatics in mouse airways and lungs under normal conditions and in models of disease. Materials and methods are described for immunohistochemical staining of lymphatics in whole mounts of the mouse trachea and 200-μm sections of mouse lung. Also described are mouse models in which lymphatics proliferate in the lung, blocking antibodies for preventing lymphatic growth, methods for fixing mouse lungs by vascular perfusion, and techniques for staining, visualizing, and analyzing lymphatic endothelial cells and other cells in the lung. These methods provide the opportunity to learn as much about lymphatics in the lung as in other organs.

Solid tumours vary in sensitivity to the vascular disrupting agent combretastatin A-4 3-O-phosphate (CA4P), but underlying factors are poorly understood. The signaling sphingolipid, sphingosine-1-phosphate (S1P), promotes vascular barrier integrity by promoting assembly of VE-cadherin/β-catenin complexes. We tested the hypothesis that tumour pre-treatment with S1P would render tumours less susceptible to CA4P. S1P (1μM) pretreatment attenuated an increase in endothelial cell (HUVEC) monolayer permeability induced by 10μM CA4P. Intravenously administered S1P (8mg/kg/hr for 20 minutes then 2mg/kg/hr for 40 minutes), reduced CA4P-induced (30mg/kg) blood flow shut-down in fibrosarcoma tumours in SCID mice (n≥7 per group), as measured by tumour retention of an intravenously administered fluorescent lectin. A trend towards in vivo protection was also found using laser Doppler flowmetry. Immunohistochemical staining of tumours ex vivo revealed disrupted patterns of VE-cadherin in vasculature of mice treated with CA4P, which were decreased by pretreatment with S1P. S1P treatment also stabilized N-cadherin junctions between endothelial cells and smooth muscle cells in culture, and stabilized tubulin filaments in HUVEC monolayers. We conclude that the rapid shutdown of tumour microvasculature by CA4P is due in part to disruption of adherens junctions and that S1P has a protective effect on both adherens junctions and the endothelial cell cytoskeleton.

Chylous pleural effusion (chylothorax) frequently accompanies lymphatic vessel malformations and other conditions with lymphatic defects. Although retrograde flow of chyle from the thoracic duct is considered a potential mechanism underlying chylothorax in patients and mouse models, the path chyle takes to reach the thoracic cavity is unclear. Herein, we use a novel transgenic mouse model, where doxycycline-induced overexpression of vascular endothelial growth factor (VEGF)-C was driven by the adipocyte-specific promoter adiponectin (ADN), to determine how chylothorax forms. Surprisingly, 100% of adult ADN-VEGF-C mice developed chylothorax within 7 days. Rapid, consistent appearance of chylothorax enabled us to examine the step-by-step development in otherwise normal adult mice. Dynamic imaging with a fluorescent tracer revealed that lymph in the thoracic duct of these mice could enter the thoracic cavity by retrograde flow into enlarged paravertebral lymphatics and subpleural lymphatic plexuses that had incompetent lymphatic valves. Pleural mesothelium overlying the lymphatic plexuses underwent exfoliation that increased during doxycycline exposure. Together, the findings indicate that chylothorax in ADN-VEGF-C mice results from retrograde flow of chyle from the thoracic duct into lymphatic tributaries with defective valves. Chyle extravasates from these plexuses and enters the thoracic cavity through exfoliated regions of the pleural mesothelium.

Lymphatic malformations are serious but poorly understood conditions that present therapeutic challenges. The goal of this study was to compare strategies for inducing regression of abnormal lymphatics and explore underlying mechanisms. CCSP-rtTA/tetO-VEGF-C mice, in which doxycycline regulates VEGF-C expression in the airway epithelium, were used as a model of pulmonary lymphangiectasia. After doxycycline was stopped, VEGF-C expression returned to normal, but lymphangiectasia persisted for at least 9 months. Inhibition of VEGFR-2/VEGFR-3 signaling, Notch, β-adrenergic receptors, or autophagy and antiinflammatory steroids had no noticeable effect on the amount or severity of lymphangiectasia. However, rapamycin inhibition of mTOR reduced lymphangiectasia by 76% within 7 days without affecting normal lymphatics. Efficacy of rapamycin was not increased by coadministration with the other agents. In prevention trials, rapamycin suppressed VEGF-C-driven mTOR phosphorylation and lymphatic endothelial cell sprouting and proliferation. However, in reversal trials, no lymphatic endothelial cell proliferation was present to block in established lymphangiectasia, and rapamycin did not increase caspase-dependent apoptosis. However, rapamycin potently suppressed Prox1 and VEGFR-3. These experiments revealed that lymphangiectasia is remarkably resistant to regression but is responsive to rapamycin, which rapidly reduces and normalizes the abnormal lymphatics without affecting normal lymphatics.

The angiopoietin/Tie (ANG/Tie) receptor system controls developmental and tumor angiogenesis, inflammatory vascular remodeling, and vessel leakage. ANG1 is a Tie2 agonist that promotes vascular stabilization in inflammation and sepsis, whereas ANG2 is a context-dependent Tie2 agonist or antagonist. A limited understanding of ANG signaling mechanisms and the orphan receptor Tie1 has hindered development of ANG/Tie-targeted therapeutics. Here, we determined that both ANG1 and ANG2 binding to Tie2 increases Tie1-Tie2 interactions in a β1 integrin-dependent manner and that Tie1 regulates ANG-induced Tie2 trafficking in endothelial cells. Endothelial Tie1 was essential for the agonist activity of ANG1 and autocrine ANG2. Deletion of endothelial Tie1 in mice reduced Tie2 phosphorylation and downstream Akt activation, increased FOXO1 nuclear localization and transcriptional activation, and prevented ANG1- and ANG2-induced capillary-to-venous remodeling. However, in acute endotoxemia, the Tie1 ectodomain that is responsible for interaction with Tie2 was rapidly cleaved, ANG1 agonist activity was decreased, and autocrine ANG2 agonist activity was lost, which led to suppression of Tie2 signaling. Tie1 cleavage also occurred in patients with hantavirus infection. These results support a model in which Tie1 directly interacts with Tie2 to promote ANG-induced vascular responses under noninflammatory conditions, whereas in inflammation, Tie1 cleavage contributes to loss of ANG2 agonist activity and vascular stability.

Angiopoietin-2 (ANG2) regulates blood vessel remodeling in many pathological conditions through differential effects on Tie2 signaling. While ANG2 competes with ANG1 to inhibit Tie2, it can paradoxically also promote Tie2 phosphorylation (p-Tie2). A related paradox is that both inactivation and overactivation of Tie2 can result in vascular remodeling. Here, we reconciled these opposing actions of ANG2 by manipulating conditions that govern its actions in the vasculature. ANG2 drove vascular remodeling during Mycoplasma pulmonis infection by acting as a Tie2 antagonist, which led to p-Tie2 suppression, forkhead box O1 (FOXO1) activation, increased ANG2 expression, and vessel leakiness. These changes were exaggerated by anti-Tie2 antibody, inhibition of PI3K signaling, or ANG2 overexpression and were reduced by anti-ANG2 antibody or exogenous ANG1. In contrast, under pathogen-free conditions, ANG2 drove vascular remodeling by acting as an agonist, promoting high p-Tie2, low FOXO1 activation, and no leakage. Tie1 activation was strong under pathogen-free conditions, but infection or TNF-α led to Tie1 inactivation by ectodomain cleavage and promoted the Tie2 antagonist action of ANG2. Together, these data indicate that ANG2 activation of Tie2 supports stable enlargement of normal nonleaky vessels, but reduction of Tie1 in inflammation leads to ANG2 antagonism of Tie2 and initiates a positive feedback loop wherein FOXO1-driven ANG2 expression promotes vascular remodeling and leakage.

Polycystic kidney diseases (PKD) are genetic disorders characterized by progressive epithelial cyst growth leading to destruction of normally functioning renal tissue. Current therapies have focused on the cyst epithelium, and little is known about how the blood and lymphatic microvasculature modulates cystogenesis. Hypomorphic Pkd1(nl/nl) mice were examined, showing that cystogenesis was associated with a disorganized pericystic network of vessels expressing platelet/endothelial cell adhesion molecule 1 and vascular endothelial growth factor receptor 3 (VEGFR3). The major ligand for VEGFR3 is VEGFC, and there were lower levels of Vegfc mRNA within the kidneys during the early stages of cystogenesis in 7-day-old Pkd1(nl/nl) mice. Seven-day-old mice were treated with exogenous VEGFC for 2 weeks on the premise that this would remodel both the VEGFR3(+) pericystic vascular network and larger renal lymphatics that may also affect the severity of PKD. Treatment with VEGFC enhanced VEGFR3 phosphorylation in the kidney, normalized the pattern of the pericystic network of vessels, and widened the large lymphatics in Pkd1(nl/nl) mice. These effects were associated with significant reductions in cystic disease, BUN and serum creatinine levels. Furthermore, VEGFC administration reduced M2 macrophage pericystic infiltrate, which has been implicated in the progression of PKD. VEGFC administration also improved cystic disease in Cys1(cpk/cpk) mice, a model of autosomal recessive PKD, leading to a modest but significant increase in lifespan. Overall, this study highlights VEGFC as a potential new treatment for some aspects of PKD, with the possibility for synergy with current epithelially targeted approaches.

The present study sought to determine the anti-tumor effects of OXA-11, a potent, novel small-molecule amino pyrimidine inhibitor (1.2 pM biochemical IC(50)) of focal adhesion kinase (FAK). In studies of cancer cell lines, OXA-11 inhibited FAK phosphorylation at phospho-tyrosine 397 with a mechanistic IC(50) of 1 nM in TOV21G tumor cells, which translated into functional suppression of proliferation in 3-dimensional culture with an EC(50) of 9 nM. Studies of OXA-11 activity in TOV21G tumor-cell xenografts in mice revealed a pharmacodynamic EC(50) of 1.8 nM, indicative of mechanistic inhibition of pFAK [Y397] in these tumors. OXA-11 inhibited TOV21G tumor growth in a dose-dependent manner and also potentiated effects of cisplatin on tumor cell proliferation and apoptosis in vitro and on tumor growth in mice. Studies of pancreatic neuroendocrine tumors in RIP-Tag2 transgenic mice revealed OXA-11 suppression of pFAK [Y397] and pFAK [Y861] in tumors and liver. OXA-11 given daily from age 14 to 17 weeks reduced tumor vascularity, invasion, and when given together with the anti-VEGFR-2 antibody DC101 reduced the incidence, abundance, and size of liver metastases. Liver micrometastases were found in 100 % of mice treated with vehicle, 84 % of mice treated with OXA-11, and 79 % of mice treated with DC101 (19-24 mice per group). In contrast, liver micrometastases were found in only 52 % of 21 mice treated with OXA-11 plus DC101, and those present were significantly smaller and less numerous. Together, these findings indicate that OXA-11 is a potent and selective inhibitor of FAK phosphorylation in vitro and in vivo. OXA-11 slows tumor growth, potentiates the anti-tumor actions of cisplatin and--when combined with VEGFR-2 blockade--reduces metastasis of pancreatic neuroendocrine tumors in RIP-Tag2 mice.

Remodeling of blood vessels and lymphatics are prominent features of sustained inflammation. Angiopoietin-2 (Ang2)/Tie2 receptor signaling and tumor necrosis factor-α (TNF)/TNF receptor signaling are known to contribute to these changes in airway inflammation after Mycoplasma pulmonis infection in mice. We determined whether Ang2 and TNF are both essential for the remodeling on blood vessels and lymphatics, and thereby influence the actions of one another. Their respective contributions to the initial stage of vascular remodeling and sprouting lymphangiogenesis were examined by comparing the effects of function-blocking antibodies to Ang2 or TNF, given individually or together during the first week after infection. As indices of efficacy, vascular enlargement, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic vessel sprouting were assessed. Inhibition of Ang2 or TNF alone reduced the remodeling of blood vessels and lymphatics, but inhibition of both together completely prevented these changes. Genome-wide analysis of changes in gene expression revealed synergistic actions of the antibody combination over a broad range of genes and signaling pathways involved in inflammatory responses. These findings demonstrate that Ang2 and TNF are essential and synergistic drivers of remodeling of blood vessels and lymphatics during the initial stage of inflammation after infection. Inhibition of Ang2 and TNF together results in widespread suppression of the inflammatory response.

Sphingosine-1-phosphate, generated by sphingosine kinases (Sphk1 and 2), acts at its receptor S1Pr1 to maintain vascular homeostasis. The gradient of S1P concentration (low micromolar in plasma vs. low nanomolar in tissue) suggests two non-mutually exclusive models via which S1P maintains the endothelial barrier: 1) plasma S1P communicates continuously with the endothelium to maintain vascular integrity and regulate vascular leak, or 2) compartmentalization of S1P and S1Pr1 enables sensing of plasma extravasation and a dynamic response to leak. To distinguish between these models, we examined endothelial responses to apical vs. basolateral application of S1P in vitro and S1Pr1 subcellular localization in vivo. Addition of S1P to the upper (apical) chamber of transwells containing EA.hy926 endothelial monolayers triggered ERK activation. Surprisingly, addition of these agonists to the lower (basolateral) chamber was without effect. Similar results were obtained with the S1Pr1 agonist SEW2871, but the PAR1 agonist SFLLRN triggered ERK activation from either chamber. By immunofluorescence staining and surface biotinylation of EA.hy926 cells, S1Pr1 protein was present predominantly on the apical surface. Similarly, staining of mouse trachea microvessels revealed S1Pr1 on CD31-positive membranes apical, but not basal, to the endothelial cell nucleus. S1Pr1 staining co-localized with the apical membrane marker podocalyxin but not the basal marker β1-integrin. The level of S1Pr1 protein in endothelial cells in mice lacking plasma S1P (Sphk1 flox/-; Sphk2 -/-; Mx-1 cre) was twice that in littermate controls. Deletion of S1Pr1 from the endothelium (S1Pr1 flox/-; Cdh5 Cre) resulted in a 6-fold increase in vascular permeability compared to littermate controls. Preliminary data suggests that S1Pr1 inactivation alters the phosphorylation state of VE-cadherin. These results suggest that S1Pr1 is polarized to the luminal membrane of endothelial cells where it is continuously activated but only partially downregulated by plasma S1P and that ongoing activation of S1Pr1 by S1P in plasma is required to maintain endothelial barrier function.

Hepatocyte growth factor (HGF) promotes lung epithelial repair after injury. Because prior studies established that human neutrophil proteases inactivate HGF in vitro, we predicted that HGF levels decrease in lungs infiltrated with neutrophils and that injury is less severe in lungs lacking HGF-inactivating proteases. After establishing that mouse neutrophil elastase cleaves mouse HGF in vitro, we tested our predictions in vivo by examining lung pathology and HGF in mice infected with Mycoplasma pulmonis, which causes neutrophilic tracheobronchitis and pneumonia. Unexpectedly, pneumonia severity was similar in wild type and dipeptidylpeptidase I-deficient (Dppi-/-) mice lacking neutrophil serine protease activity. To assess how this finding related to our prediction that Dppi-activated proteases regulate HGF levels, we measured HGF in serum, bronchoalveolar lavage fluid, and lung tissue from Dppi(+/+) and Dppi(-/-) mice. Contrary to prediction, HGF levels were higher in lavage fluid from infected mice. However, serum and tissue concentrations were not different in infected and uninfected mice, and HGF lung transcript levels did not change. Increased HGF correlated with increased albumin in lavage fluid from infected mice, and immunostaining failed to detect increased lung tissue expression of HGF in infected mice. These findings are consistent with trans-alveolar flux rather than local production as the source of increased HGF in lavage fluid. However, levels of intact HGF from infected mice, normalized for albumin concentration, were two-fold higher in Dppi(-/-) versus Dppi(+/+) lavage fluid, suggesting regulation by Dppi-activated proteases. Consistent with the presence of active HGF, increased expression of activated receptor c-Met was observed in infected tissues. These data suggest that HGF entering alveoli from the bloodstream during pneumonia compensates for destruction by Dppi-activated inflammatory proteases to allow HGF to contribute to epithelial repair.

Mast cells (MC) and myeloid dendritic cells (DC) act proximally in detecting and processing antigens and immune insults. We sought to understand their comparative dynamic behavior with respect to the airway epithelium in the steady state and in response to an allergic stimulus in mouse trachea. We devised methods to label MC in living trachea and to demonstrate that MC and DC occupy distinct layers of the tracheal mucosa, with DC being closer to the lumen. DC numbers doubled after allergen challenge, but MC numbers remained stable. MC and DC migrated minimally in either steady state or allergen-challenge conditions, and their interactions with one another appeared to be stochastic and relatively infrequent. While DC, unlike MC, exhibited probing behaviors involving dendrites, these projections did not cross the epithelium into the airway lumen. MC typically were located too far from the epithelial surface to contact the tracheal lumen. However, MC had protrusions toward and into blood vessels, likely to load with IgE. Thus, DC and MC occupy distinct niches and engage in sessile surveillance in the mouse trachea. Little or no access of these cell types to the airway lumen suggests that trans-epithelial transport of proteins in the steady state would be required for them to access luminal antigens.