Resolving the fibrotic niche of human liver cirrhosis at single-cell level.

Liver cirrhosis is a major cause of death worldwide and is characterized by extensive fibrosis. There are currently no effective antifibrotic therapies available. To obtain a better understanding of the cellular and molecular mechanisms involved in disease pathogenesis and enable the discovery of therapeutic targets, here we profile the transcriptomes of more than 100,000 single human cells, yielding molecular definitions for non-parenchymal cell types that are found in healthy and cirrhotic human liver. We identify a scar-associated TREM2+CD9+ subpopulation of macrophages, which expands in liver fibrosis, differentiates from circulating monocytes and is pro-fibrogenic. We also define ACKR1+ and PLVAP+ endothelial cells that expand in cirrhosis, are topographically restricted to the fibrotic niche and enhance the transmigration of leucocytes. Multi-lineage modelling of ligand and receptor interactions between the scar-associated macrophages, endothelial cells and PDGFRα+ collagen-producing mesenchymal cells reveals intra-scar activity of several pro-fibrogenic pathways including TNFRSF12A, PDGFR and NOTCH signalling. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ fibrosis at a single-cell level, and provides a conceptual framework for the discovery of rational therapeutic targets in liver cirrhosis.

Identification and angiogenic role of the novel tumor endothelial marker CLEC14A.

Tumor endothelial markers (TEMs) that are highly expressed in human tumor vasculature compared with vasculature in normal tissue hold clear therapeutic potential. We report that the C-type lectin CLEC14A is a novel TEM. Immunohistochemical and immunofluorescence staining of tissue arrays has shown that CLEC14A is strongly expressed in tumor vasculature when compared with vessels in normal tissue. CLEC14A overexpression in tumor vessels was seen in a wide range of solid tumor types. Functional studies showed that CLEC14A induces filopodia and facilitates endothelial migration, tube formation and vascular development in zebrafish that is, CLEC14A regulates pro-angiogenic phenotypes. CLEC14A antisera inhibited cell migration and tube formation, suggesting that anti-CLEC14A antibodies may have anti-angiogenic activity. Finally, in endothelial cultures, expression of CLEC14A increased at low shear stress, and we hypothesize that low shear stress due to poor blood flow in the disorganized tumor vasculature induces expression of CLEC14A on tumor vessels and pro-angiogenic phenotypes.

Comparison of the pro-inflammatory potential of monocytes from healthy adults and those with peripheral arterial disease using an in vitro culture model.

We adapted a monocyte:endothelial cell co-culture model to investigate the pro-inflammatory potential of monocytes from patients with peripheral arterial disease (PAD). Isolated monocytes were cultured with human umbilical vein endothelial cells (HUVEC) for 24h, after which the ability of the HUVEC to recruit flowing neutrophils was tested. Development of a usable protocol required comparisons of primary HUVEC with cells that had been passaged and/or frozen and thawed, evaluation of optimal culture media and comparison of monocytes from freshly drawn and stored blood. We found, for instance, that expansion of HUVEC was assisted by inclusion of hydrocortisone, but this agent was withdrawn before the test phase because it reduced responses of HUVEC. Using the optimal practical protocol, we found great variation in the ability of monocytes from different donors to cause neutrophil adhesion. Slightly more ( approximately 20%) monocytes from patients with PAD adhered to HUVEC than monocytes from healthy controls, and the monocytes from PAD patients induced approximately 70% greater subsequent adhesion of neutrophils. Thus, we developed a functional model of inflammatory potential usable in clinically-related studies and found that patients with PAD had circulating monocytes with greater than normal ability to activate endothelial cells.

Antineutrophil cytoplasmic antibodies stabilize adhesion and promote migration of flowing neutrophils on endothelial cells.

OBJECTIVE: Recruitment of neutrophils to sites of inflammation requires coordinated regulation of their capture, activation, and migration on vascular endothelium. This study examines whether exposure of neutrophils to antineutrophil cytoplasmic antibodies (ANCAs) can disrupt this sequence of events. METHODS: Isolated human neutrophils were perfused in the presence or absence of ANCA-positive IgG over endothelial cells that had been activated with either 2 units/ml or 100 units/ml of tumor necrosis factor alpha (TNFalpha) for 4 hours. RESULTS: When endothelial cells were activated with 100 units/ml of TNFalpha, neutrophils were captured from flow, a small proportion of adherent cells rolled, and the majority transmigrated through the endothelial cell monolayer. When neutrophils were treated with ANCA IgG immediately before, 5 minutes before, or 15 minutes before perfusion, none rolled on contact with the endothelium, but the majority still transmigrated. When endothelial cells were activated with 2 units/ml of TNFalpha, the majority of untreated adherent neutrophils rolled, a few transmigrated, and the number that attached decreased with time during washout. In contrast, when neutrophils were pretreated with ANCA IgG just before perfusion, adhesion was stabilized, and the number of neutrophils that transmigrated was increased 10-fold. Priming of the neutrophils with TNFalpha before the addition of ANCA further increased the stability of neutrophil binding, but did not significantly increase transmigration. CONCLUSION: Rather than frustrating the transmigration process, ANCAs promoted the migration of neutrophils through the endothelium. That the effect was evident at a relatively low level of endothelial activation suggests that ANCAs may potentiate the early vasculitic lesion and promote tissue damage and recruitment of other proinflammatory cells.

Differential ability of exogenous chemotactic agents to disrupt transendothelial migration of flowing neutrophils.

Neutrophils migrate through endothelium using an ordered sequence of adhesive interactions and activating signals. To investigate the consequences of disruption of this sequence, we characterized adhesion and migration of neutrophils perfused over HUVEC that had been treated with TNF-alpha for 4 h and evaluated changes caused by exogenously added chemotactic agents. When HUVEC were treated with 2 U/ml TNF, flowing neutrophils adhered, with the majority rolling and relatively few migrating through the monolayer. If fMLP, IL-8, zymosan-activated plasma (a source of activated complement factor C5a), epithelial cell-derived neutrophil-activating peptide (ENA-78), or growth-regulating oncogene, GRO-alpha, was perfused over these neutrophils, they stopped rolling and rapidly migrated over the monolayer, but did not penetrate it. When HUVEC were treated with 100 U/ml TNF, the majority of adherent neutrophils transmigrated. If neutrophils were treated with fMLP, IL-8, C5a, ENA-78, or GRO-alpha just before perfusion over this HUVEC, transmigration, but not adhesion, was abolished. However, when platelet-activating factor was used to activate neutrophils, migration through HUVEC treated with 100 U/ml TNF was not impaired, and migration through HUVEC treated with 2 U/ml TNF was actually increased. Transmigration required ligation of CXC chemokine receptor-2 on neutrophils, and differential desensitization of this receptor (e.g., by fMLP but not platelet-activating factor) may explain the pattern of disruption of migration. Thus, transmigration may require presentation of the correct activators in the correct sequence, and inappropriate activation (e.g., by systemic activators) could cause pathological accumulation of neutrophils in the vessel lumen.

Synergistic interactions between ampakines and antipsychotic drugs.

Tests were made for interactions between antipsychotic drugs and compounds that enhance synaptic currents mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptors ("ampakines"). Typical and atypical antipsychotic drugs decreased methamphetamine-induced hyperactivity in rats; the effects of near or even subthreshold doses of the antipsychotics were greatly enhanced by the ampakines. Interactions between the ampakine CX516 and low doses of different antipsychotics were generally additive and often synergistic. The ampakine did not exacerbate neuroleptic-induced catalepsy, indicating that the interaction between the different pharmacological classes was selective. These results suggest that positive modulators of cortical glutamatergic systems may be useful adjuncts in treating schizophrenia.

Kinetics of the different steps during neutrophil migration through cultured endothelial monolayers treated with tumour necrosis factor-alpha.

To enable a better understanding of the regulation of neutrophil migration, we investigated the kinetics of adhesion and migration over, through and under endothelial monolayers. Neutrophils were perfused over human umbilical vein endothelial cells (HUVEC) which had been treated with tumour necrosis factor-alpha (TNF; 2-1,000 U/ml) for 4 h. Videomicroscopy showed that transendothelial migration was complete within about 5 min of completion of perfusion of a bolus of neutrophils. Separate populations of adherent cells could then be observed, either rolling, migrating over the surface of the HUVEC or migrating underneath, at different characteristic speeds. Increasing concentration of TNF had little effect on the kinetics of migration, but shifted the balance from rolling adhesion to transendothelial migration. When individual neutrophils were followed from the moment they bound to HUVEC treated with 100 U/ml TNF, we found that approximately 40% immobilised essentially immediately on contact, while approximately 40% immobilised after rolling for varying periods (average 26 s) and approximately 20% rolled continuously. Most of the immobilised cells went on to migrate through the monolayer after spending 20-200 s migrating on top, and took about 60 s to pass through. Overall, the time from first binding to completion of transmigration averaged 152 s (range approximately 60-240 s). Interestingly, neutrophils moved relatively slowly on top of the monolayer (about 8 microm/min) but more rapidly underneath (about 16 microm/min). We suggest that the different stages during neutrophil transmigration have characteristic kinetics with separate control mechanisms, which critically influence the efficiency and rate of clearance from the vasculature.

Attenuation of ozone-induced airway permeability in rats by pretreatment with cyclophosphamide, FPL 55712, and indomethacin.

Exposure of rats to ozone (O3) produces an increase in airway permeability and a concomitant influx of polymorphonuclear leukocytes in the lung. These observations raise the possibility that the inflammatory cells play a role in the cellular injury and increased airway permeability after O3 exposure. This study was therefore designed to determine if the inflammatory cells or their products are essential for the O3 effect. In a series of experiments, rats were rendered leukopenic with cyclophosphamide, treated with leukotriene B4 (LTB4), or with the inhibitors of lipoxygenase or cyclooxygenase products of arachidonic acid, followed by exposure to O3. A 2-h exposure to 0.8 ppm O3 caused a significant increase in the flux of proteins and albumin in bronchoalveolar lavage (BAL) and elevated the transport of 99mTc-diethylenetriaminepentaacetate (99mTc-DTPA) from trachea to blood. The treatment with cyclophosphamide caused a significant reduction in the circulating and pulmonary leukocytes and prevented an increase in tracheal mucosal permeability to 99mTc-DTPA and the protein and albumin flux in BAL. While the intratracheal instillation of LTB4 did not affect the permeability, tracheal permeability and albumin levels in BAL in rats treated with LTD4 antagonist FPL 55712 and exposed to O3 were lower than in the untreated O3-exposed rats. Pretreatment with indomethacin also prevented the O3 effects, as reflected by the decreased protein and albumin flux in BAL and 99mTc-DTPA transport from trachea to blood. These data show a reduction in the effect of O3 by agents that affect leukocytes or their products. The results support a mechanism of increased permeability that is dependent upon inflammatory cells and their products.