Search

Sickle cell disease (SCD) is associated with hemolysis, vascular inflammation, and organ damage. Affected patients experience chronic painful vaso-occlusive events requiring hospitalization. Hypoxia-induced polymerization of sickle hemoglobin S (HbS) contributes to sickling of red blood cells (RBCs) and disease pathophysiology. Dilution of HbS with nonsickling hemoglobin or hemoglobin with increased oxygen affinity, such as fetal hemoglobin or HbS bound to aromatic aldehydes, is clinically beneficial in decreasing polymerization. We investigated a novel alternate approach to modify HbS and decrease polymerization by inhibiting methionine aminopeptidase 2 (MetAP2), which cleaves the initiator methionine (iMet) from Val1 of α-globin and βS-globin. Kinetic studies with MetAP2 show that βS-globin is a fivefold better substrate than α-globin. Knockdown of MetAP2 in human umbilical cord blood–derived erythroid progenitor 2 cells shows more extensive modification of α-globin than β-globin, consistent with kinetic data. Treatment of human erythroid cells in vitro or Townes SCD mice in vivo with selective MetAP2 inhibitors extensively modifies both globins with N-terminal iMet and acetylated iMet. HbS modification by MetAP2 inhibition increases oxygen affinity, as measured by decreased oxygen tension at which hemoglobin is 50% saturated. Acetyl-iMet modification on βS-globin delays HbS polymerization under hypoxia. MetAP2 inhibitor–treated Townes mice reach 50% total HbS modification, significantly increasing the affinity of RBCs for oxygen, increasing whole blood single-cell RBC oxygen saturation, and decreasing fractional flow velocity losses in blood rheology under decreased oxygen pressures. Crystal structures of modified HbS variants show stabilization of the nonpolymerizing high O2–affinity R2 state, explaining modified HbS antisickling activity. Further study of MetAP2 inhibition as a potential therapeutic target for SCD is warranted.

There is an emerging interest in the diverse functions of neutrophil extracellular traps (NETs) in a variety of disease settings. However, data on circulating NETs rely largely upon surrogate NET markers such as cell-free DNA, nucleosomes, and NET-associated enzymes. Citrullination of histone H3 by peptidyl arginine deiminase 4 (PAD4) is central for NET formation, and citrullinated histone H3 (H3Cit) is considered a NET-specific biomarker. We therefore aimed to optimize and validate a new enzyme-linked immunosorbent assay (ELISA) to quantify the levels of H3Cit in human plasma. A standard curve made of in vitro PAD4-citrullinated histones H3 allows for the quantification of H3Cit in plasma using an anti-histone antibody as capture antibody and an anti-histone H3 citrulline antibody for detection. The assay was evaluated for linearity, stability, specificity, and precision on plasma samples obtained from a human model of inflammation before and after lipopolysaccharide injection. The results revealed linearity and high specificity demonstrated by the inability of detecting non-citrullinated histone H3. Coefficients of variation for intra- and inter-assay variability ranged from 2.1 to 5.1% and from 5.8 to 13.5%, respectively, allowing for a high precision. Furthermore, our results support an inflammatory induction of a systemic NET burden by showing, for the first time, clear intra-individual elevations of plasma H3Cit in a human model of lipopolysaccharide-induced inflammation. Taken together, our work demonstrates the development of a new method for the quantification of H3Cit by ELISA that can reliably be used for the detection of NETs in human plasma.

Sickle cell disease (SCD) is a hereditary disorder occurring due to a mutation in the β- globin gene resulting in hemoglobin polymerization and sickling of red blood cells that drives an array of severe pathophysiologies. SCD patients with hereditary persistence of fetal hemoglobin mutations show amelioration of disease symptoms. HRI is a heme sensing eIF2α kinase belonging to the integrated stress response pathway, primarily regulating the hemoglobin synthesis in RBCs. Under low levels of heme, HRI undergoes auto-phosphorylation and subsequently phosphorylates its substrate eIF2α. This impedes the global protein translation and selectively activates ATF4 translation thereby initiating a transcriptional stress response. HRI protein deletion has been shown to induce fetal hemoglobin (HbF) in vitro by reducing BCL11A levels (Grevet et al., 2018). It has also been shown that ATF4 binds to enhancer region of BCL11A promoting its expression, revealing a direct regulation of HRI from eIF2α to ATF4 to BCL11A to γ-globin (Huang P, 2020). As it is well-established that protein kinases can exert dual kinase and scaffold functions, we explored whether the regulation of HRI on HbF is driven by its catalytic or non-catalytic activity. We generated HRI kinase dead (KD) mutant HUDEP-2 cells, with a mutation (K196R) in HRI ATP binding site that is predicted to result in loss of its auto-phosphorylation (Bauer B. N., 2001). HRI KD mutant HUDEP-2 was created using CRISPR Cas9 and homologous recombination approach, followed by clonal selection that yielded two homozygous clones, referred as KD clones. To further compare the scaffold and kinase function of HRI, we created HRI knockout (KO) in HUDEP-2 cell line using CRISPR Cas9 approach. We isolated clonal populations with 3 clones each with heterozygous and homozygous editing. Western analysis of KD clones showed that HRI protein levels remain unchanged. About 50% and a 100% loss in HRI protein level was observed in heterozygous and homozygous KO clones respectively. To confirm the loss in kinase activity of HRI, we differentiated KD clones for 7 days in vitro and subjected them to iron depleted conditions. As expected, HRI WT cells under iron depleted conditions showed robust activation in the p-eIF2α levels indicative of increase in HRI activity. KD clones lacked increase in p-eIF2α levels under these conditions confirming the loss of kinase activity of HRI in KD clones. HRI KD and KO clones were characterized to evaluate the HbF regulation by differentiating in vitro for 7-8 days. KD clones showed 4-5-fold induction in HbF levels measured by flow cytometry. Heterozygous and homozygous KO clones showed an equally strong activation in HbF levels. KD and KO clones showed downregulation in BCL11A and ATF4 protein levels. These results indicate that HRI regulates HbF as a function of its catalytic activity. We further employed RNA-seq to study the global transcriptomics in HRI WT and KD mutant clones at day 0 and day 8, under steady state and iron depleted conditions. RNA-seq confirmed the strong activation of HBG1/2 levels and robust silencing of BCL11A levels in KD clones at day 8. A weaker induction in HBG1/2 levels and minor reduction on BCL11A levels were observed at day 0. These results indicate that HRI may play a role in HbF silencing in a stage specific manner. HRI WT cells under iron depleted condition showed activation of ATF4 pathway, indicative of activation of HRI stress pathway. As expected, HRI KD population showed no regulation of ATF4 pathway under iron depleted conditions. Interestingly, we did not observe any changes in the BCL11A gene expression in WT cells under iron depleted conditions. Further evaluation needs to be carried out in order to understand the regulation of BCL11A by ATF4 under low iron mediated stress. Fewer gene changes were observed in KD populations under iron depleted conditions indicative of HRI playing a vital role as a heme sensor. Overall, our results provide evidence to support that HRI regulates HbF though its catalytic activity and demonstrates its role as heme sensor in HUDEP-2 cells. Disclosures Gupta: Sanofi: Current Employment. Hara:Sanofi: Current Employment. Lessard:Sanofi: Current Employment. Sturtevant:Sanofi: Current Employment. Krishnamoorthy:Sanofi: Current Employment. Demers:Sanofi: Current Employment. Hicks:Sanofi: Current Employment.

Large elevations of high sensitive Troponin T (hsTnT) in ischemic stroke patients is associated with a poor outcome. In a pilot study we found a high prevalence of malignancies among these patients. Since neutrophil extracellular traps (NETs) have been linked to cancer-associated thrombosis, we hypothesized that the concomitant cerebral and myocardial ischemia could be the result of a NET-induced hypercoagulable state.Clinical assessments, plasma analyses and autopsies with histopathology (in cases of in-hospital mortality) were performed on ischemic stroke patients with high elevations of hsTnT (N=12) and normal hsTnT (N=19).Patients with hsTnT elevation had an unexpectedly higher prevalence of cancer (p=0.002), half of which were diagnosed post-mortem. Autopsies of these patients revealed widespread myocardial, cerebral and pulmonary microthrombosis with H3Cit in thrombi. A pro-coagulant state and an increase of the NET specific marker citrullinated histone H3 (H3Cit) was found in plasma of patients with elevated hsTnT compared to patients with normal levels (p0.001). Plasma analyses in cancer patients showed even higher H3Cit levels (p0.001), and an increase in granulocyte colony-stimulating factor, known to prime neutrophils towards NETosis. H3Cit correlated positively with thrombin-antithrombin complex (p=0.004) and soluble P-selectin (p0.001), further linking NETosis to the pro-thrombotic state.The high prevalence of known or occult cancer in our study suggests that cancer-associated arterial microthrombosis may be underestimated. By linking the thrombosis to NETs, we suggest markers of NETosis that could aid in revealing cancer in arterial microthrombosis as well as arterial microthrombosis in cancer.

Thrombotic microangiopathy (TMA) is a life-threatening condition that affects some, but not all, recipients of vascular endothelial growth factor (VEGF) inhibitors given as part of chemotherapy. TMA is also a complication of preeclampsia, a disease characterized by excess production of the VEGF-scavenging soluble VEGF receptor 1 (soluble fms-like tyrosine kinase 1; sFlt-1). Risk factors for VEGF inhibitor-related TMA remain unknown. We hypothesized that deficiency of the VWF-cleaving ADAMTS13 endopeptidase contributes to the development of VEGF inhibitor-related TMA. ADAMTS13(-/-) mice overexpressing sFlt-1 presented all hallmarks of TMA, including thrombocytopenia, schistocytosis, anemia, and VWF-positive microthrombi in multiple organs. Similar to VEGF inhibitor-related TMA in humans, these mice exhibited severely impaired kidney function and hypertension. In contrast, wild-type mice overexpressing sFlt-1 developed modest hypertension but no other features of TMA. Recombinant ADAMTS13 therapy ameliorated all symptoms of TMA in ADAMTS13(-/-) mice overexpressing sFlt-1 and normalized BP in wild-type mice. ADAMTS13 activity may thus be a critical determinant for the development of TMA secondary to VEGF inhibition. Administration of recombinant ADAMTS13 may serve as a therapeutic approach to treat or prevent thrombotic complications of VEGF inhibition.

Sickle cell disease (SCD) is characterized by acute and repetitive vaso-occlusive crises (VOC). These crises have been hypothesized to occur when blood flow is reduced following obstruction of sickle-shaped red blood cells in the vasculature. However, it is now well established that inflammation, oxidative stress, endothelial activation and pro-coagulation in sickle cell disease patients also contribute to the formation of heterocellular aggregates that can lead to VOC (Vercellotti and Belcher, 2014). Transgenic SCD mice recapitulate the pathology of human disease in response to stimuli such as heme injection and hypoxia/reoxygenation. SCD SS Townes mice, which express human α and sickle γAβS globins, AA Townes mice expressing normal human α and normal γAβA globins and heterozygous AS mice which express only one allele of the γAβS sickle gene were used. To characterize vaso-occlusion in these mice and evaluate the efficacy of different pharmacological mechanisms, we modified the skinfold vaso-occlusion model (Kalambur et al, 2004) using fluorescent intravital microscopy to visualize blood flow occlusion following hemin injection or hypoxia/reoxygenation challenge. Dorsal skinfold chambers were implanted and24 hours post-surgery mice were injected with FITC-dextran for visualization of flowing blood vessels. Skinfold bearing mice were then subjected to hemin treatment (16 μmoles/kg) or hypoxia (7%; 1 hour)/reoxygenation (1 hour) followed by the injection of Alexa fluor 647-labeled albumin to allow quantification of occluded vessels through dual fluorescent image analysis. Following hemin injection, SS mice showed significant ~30% vaso-occlusion in comparison to AA mice with ~8%, whereas the AS mice showed an intermediate phenotype with ~20% vaso-occlusion. Hypoxia/reoxygenation challenge also resulted in significant vaso-occlusion for SS mice (~25%) whereas only 5% was observed in AA mice. Interestingly, AS mice also showed a significant amount of vaso-occlusion (~25%) similar to SS mice when challenged with hypoxia/reoxygenation. Although no sickling can be observed in an ex vivo sickling assay using AS red blood cells, an intermediate amount of free Hemoglobin (Hb) can be detected in the plasma of these mice and rolling can be observed. This suggests that these vaso-occlusive models relate more on the inflammatory and endothelial activation state and are independent of the sickling potential of the red blood cell. We then used our model with hypoxia/reoxygenation challenge to evaluate the effects of dimethyl fumarate (DMF, 15 mpk BID), an anti-P-Selectin antibody (150ug/mouse) and the covalent hemoglobin oxygen affinity modulator GBT-440 (300 mpk). As anti-inflammatory agents, DMF and Anti-P-Selectin significantly reduced vaso-occlusion in SS mice by ~60% compared to the vehicle treated mice, but GBT-440 did not inhibit vaso-occlusion at a dose where a significant reduction in p50 was observed. In conclusion, our data have shown that obstruction of blood flow in the skinfold vaso-occlusion model in SCD Townes mice reflects the vascular inflammatory state of the disease and is independent of the ex vivo capacity of red blood cell to sickle. Disclosures Sturtevant: Sanofi: Employment. Macias-Garcia:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment. van der Flier:Sanofi: Employment. Hicks:Sanofi: Employment. Demers:Sanofi: Employment.

Sickle cell disease (SCD) is a genetic disorder caused by a missense mutation in the beta-globin gene [Glu6Val] resulting in the formation of HbS (sickle hemoglobin). HbS polymerizes under low oxygen tension causing an array of severe pathophysiological complications, including vaso-occlusion, pain and hemolytic anemia. Traditionally, research efforts toward studying anti-sickling effects of various pathways and compounds in vitro have been made using sickle cell patient derived samples. However, such studies are limited based on sample availability and a large degree of phenotypic variability. Thus, a cultured cell system with a sickle mutation mimicking human sickle cell erythroid cells is a potentially useful tool that can be used to interrogate anti-sickling compound action and to study various facets of sickle cell phenotype. In this study, we generated a genetically engineered sickle mutation (GAG to GTG) in HUDEP2 cell line using CRISPR Cas9 gene editing and homology directed repair mechanism. HUDEP2 cells are immortalized human erythroid progenitor cells, obtained from the Riken Institute and were cultured as described (Kurita et al 2013). CRISPR Cas9 RNP complex with modified guide RNA along with ssODN were electroporated in HUDEP2 cells. Cells were also treated with SCR7, a NHEJ inhibitor to facilitate homology- directed repair events. Cells were sorted for clonal selection, cultured and further screened for homozygous knock-in mutations using digital droplet PCR. The mutation was further confirmed by sanger sequencing. 12 clones consisting of homozygous mutation were identified, with 6 of these clones selected for further characterization. Cells were differentiated for 7 days in culture and hemolysates were analyzed using HPLC. Heterozygote clone showed 32.3% HbS which was comparable to sickle cell trait patient derived blood samples whereas homozygous clones (ssHUDEP2) showed between 71-85% HbS. These results were further confirmed for the presence of HbS using mass spectrometry. Mutant HUDEP2 cell growth and differentiation were similar to the wild-type HUDEP2 cells. To evaluate sickling in these cells we optimized the differentiation protocol to improve the maturation of these cells by withdrawing SCF and doxycycline gradually. Hemoglobin polymerization and resulting shape change in sickle HUDEP cells was determined by subjecting mature ssHUDEP2 cells to hypoxia (1% oxygen) for 3 hours. Upon subjecting to hypoxia, ssHUDEP2 cells showed morphological changes indicative of HbS polymerization. These morphological changes were absent in wild type cells under similar conditions of hypoxia. In addition, ssHUDEP2 cells pre-treated with 25µM GBT440, a known anti-sickling compound, for 1 hour prior to hypoxia incubation elicited a reduction in the shape change associated with hypoxia, suggesting inhibition of hemoglobin polymerization and sickling. These results suggest that genetically-engineered sickle mutant HUDEP cells represent a preclinical model for development of anti-sickling compounds for the potential treatment of Sickle Cell Disease and to explore novel mechanistic pathways. Disclosures Gupta: Sanofi: Employment. Sturtevant:Sanofi: Employment. Vieira:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment. Demers:Sanofi: Employment.

Dilution of HbS with non-sickling hemoglobin or hemoglobin with increased oxygen affinity is clinically beneficial in sickle cell disease. Aldehydes, including 5-HMF, tucaresol or GBT440, modify the N-terminus of HbS by reversible covalent imine formation generating modified forms of HbS that resist polymerization under low oxygen concentrations. In contrast to reversible imine formation by aldehydes, we hypothesize that stable modification of HbS will result from N-terminal retention of the initiator methionine (iMet) and subsequent N-terminal acetylation of the iMet (acetyl-iMet). MetAP2 is the methionine aminopeptidase able to cleave iMet from Val1 on α-globin and βS-globin as the unfolded N-terminal peptides emerge from the ribosome. Enzyme kinetic studies with pure MetAP2 and N-terminal octapeptides showed that βS-globin peptide is a 5-fold better substrate than α-globin peptide. Lentiviral shRNA knock-down of MetAP2 in differentiating erythroid HUDEP cells in vitro confirmed that α-globin is more extensively modified than βS-globin, consistent with the enzyme kinetic data. Selective MetAP2 inhibitors used to treat cultured human erythroid cells (HUDEP and PBMC derived CD34+) and Townes SCD mice in vivo confirmed that both α-globin and βS-globin domains of HbS are extensively modified by N-terminal iMet and acetyl-iMet. N-terminal retention of iMet and subsequent acetylation creates a mixture of modified HbS tetramers with combined modifications on both globins. Cation exchange chromatography separated nine different modified HbS variants from unmodified HbS as identified by LCMS. Purified samples of HbS modified by N-terminal iMet and acetyl-iMet had increased oxygen affinity as measured by decreased P50. Modified HbS containing the acetyl-iMet-βS-globin were found to have delayed polymerization under complete hypoxia (sodium metabisulfite triggered hypoxia in 1.8 M phosphate). Two modified HbS variants were further purified for X-ray crystallography studies (βS-globin / iMet-α-globin and acetyl-iMet-βS-globin / iMet-α-globin). Oxyhemoglobin structures of both modified HbS variants were in the R2-state previously described in structures of aldehyde modified HbS. This R2-state stabilizes the oxygenated R-state of HbS from conversion to the deoxygenated T-state that initiates HbS polymerization in sickle RBC. Treatment by selective irreversible covalent or reversible MetAP2 inhibitors resulted in high levels of HbS modification (>75%) in cultured erythroid cells (HUDEP and CD34+ cells). Dose dependent modification of HbS was observed in Townes sickle cell mouse blood RBC in vivo with total modification of HbS approaching 50%. In whole blood ex vivo studies, modification of HbS decreased RBC sickling under hypoxia (4% O2) and significantly increased the affinity of RBC for oxygen (decreased P50). Blood samples from MetAP2 inhibitor treated mice were analyzed for single-cell O2 saturation of the RBC and for the fractional flow velocity drop in whole blood rheology under decreasing partial oxygen pressures. In blood from vehicle treated sickle mice, a low-saturation peak of deoxy-HbS was observed in 7.8% O2, in contrast to blood from MetAP2 inhibitor-treated mice where the low-saturation peak was only observed in 6.4% O2. Similarly, in an assay of O2 dependent blood flow rheology, the half-maximum fractional velocity drop occurred at 5% O2 in control blood decreasing to 2% O2 in MetAP2 inhibitor treated blood. Our studies show that MetAP2 inhibition results in retention of iMet on βS-globin and α-globin and allows further acetylation of the retained iMet to create a mixture of N-terminal modified HbS tetramers. These modified HbS variants resist polymerization and RBC sickling under conditions of low O2 by delaying HbS polymerization and increasing O2 affinity. Our data suggests that MetAP2 may warrant further study as a potential therapeutic target for sickle cell disease. Disclosures Demers: Sanofi: Employment. Sturtevant:Sanofi: Employment. Guertin:Sanofi: Employment. Gupta:Sanofi: Employment. Desai:Sanofi: Employment. Vieira:Sanofi: Employment. Hicks:Sanofi: Employment. Ismail:Sanofi: Employment. Safo:Sanofi: Consultancy, Research Funding; Virginia Commonwealth University: Patents & Royalties. Wood:Sanofi: Consultancy, Research Funding. Higgins:Sanofi: Consultancy, Research Funding. Light:Sanofi: Employment.

Background Inflammatory bowel disease (IBD) affects 1.6 million people in the United States. IBD is associated with an increased risk of thrombosis, which rises with disease activity. The pathogenesis of IBD and its increased thrombotic risk is not completely understood. Ultra large von Willebrand factor (ULVWF) multimers are secreted from activated endothelium, leading to recruitment of platelets and leukocytes. A disintegrin and metalloproteinase with thrombospondin type I repeats motif 13 (ADAMTS13) cleaves highly adhesive ULVWF into smaller, less bioactive, multimers, releasing them into circulation. Mice deficient in ADAMTS13 (ADAMTS13−/−) have heightened inflammatory and thrombotic responses. Objectives We hypothesized that upon colitis induction, ADAMTS13−/− mice would have more severe symptoms compared with wild-type (WT) mice, and rhADAMTS13 administration to mice with colitis would improve their condition. Results Dextran sodium sulfate–induced colitis was worse in ADAMTS13−/− mice than WT. ADAMTS13−/− showed increased weight loss, worse anemia, and increased clinical and histologic colitis severity, compared with WT mice. ADAMTS13−/− mice had increased VWF release, with accumulation at inflamed colonic sites. Also, the majority of mice showed one or more submucosal colonic thrombi. ADAMTS13 deficiency worsened colitis and propagated intestinal inflammation, most likely through increased platelet–leukocyte recruitment by VWF. Treatment of WT mice with rhADAMTS13 decreased colitis severity without worsening anemia. Additionally, several immune-mediated chronic murine colitis models, and inflamed colon tissue specimens from IBD patients, showed increased VWF release at inflamed sites, suggesting a generalizability of our findings. Conclusion Measuring VWF/ADAMTS13 levels could have clinical utility. When applicable, the administration of ADAMTS13, in addition to primary treatment, may improve outcomes for IBD patients.

Citrullinated histone H3 (H3Cit) is a central player in the neutrophil release of nuclear chromatin, known as neutrophil extracellular traps (NETs). NETs have been shown to elicit harmful effects on the host, and were recently proposed to promote tumor progression and spread. Here we report significant elevations of plasma H3Cit in patients with advanced cancer compared with age-matched healthy individuals. These elevations were specific to cancer patients as no increase was observed in severely ill and hospitalized patients with a higher non-malignant comorbidity. The analysis of neutrophils from cancer patients showed a higher proportion of neutrophils positive for intracellular H3Cit compared to severely ill patients. Moreover, the presence of plasma H3Cit in cancer patients strongly correlated with neutrophil activation markers neutrophil elastase (NE) and myeloperoxidase (MPO), and the inflammatory cytokines interleukin-6 and -8, known to induce NETosis. In addition, we show that high levels of circulating H3Cit strongly predicted poor clinical outcome in our cohort of cancer patients with a 2-fold increased risk for short-term mortality. Our results also corroborate the association of NE, interleukin-6 and -8 with poor clinical outcome. Taken together, our results are the first to unveil H3Cit as a potential diagnostic and prognostic blood marker associated with an exacerbated inflammatory response in patients with advanced cancer.

Prevalence of depression is higher in patients with cancer than in the general population. Sustained systemic inflammation has been associated with depressive behavior and it has been reported that depressed patients commonly display alterations in their immune system. We previously showed that cancer in mice induces a systemic environment that promotes neutrophil activation and leukocytosis. We thus hypothesized that the peripheral systemic response to a solid tumor leads to endothelial activation, which may promote inflammatory changes in the brain with behavioral consequences. Using the Lewis lung carcinoma (LLC) model, we show that tumor growth induces a progressive increase in peripheral inflammation as observed by elevated interleukin-6 (IL-6). In behavioral studies, tumor-bearing mice showed no sign of motor, coordination or short term working memory deficits as assessed by rotarod, balance-beam, and novel object recognition tests. However, there was an impairment in the grip strength test and interestingly, an anxious and despair-like phenotype in the elevated plus-maze, and tail suspension tests, respectively. Immunostaining of perfused brains revealed fibrin accumulation in the vasculature with some leakage into the parenchyma, a process known to activate endothelial cells. Taken together, our results suggest that the inflamed and prothrombotic systemic environment created by the growth of a peripherally-located solid tumor induces endothelial activation, accumulation of fibrin in the brain and astrocyte activation, perhaps leading to depressive-like behavior. ispartof: PLOS ONE vol:13 issue:11 ispartof: location:United States status: published

Wound healing is impaired in diabetes, resulting in significant morbidity and mortality. Neutrophils are the main leukocytes involved in the early phase of healing. As part of their anti-microbial defense, neutrophils form extracellular traps (NETs) by releasing decondensed chromatin lined with cytotoxic proteins. NETs, however, can also induce tissue damage. Here we show that neutrophils isolated from type 1 and type 2 diabetic humans and mice were primed to produce NETs (a process termed NETosis). Expression of peptidylarginine deiminase 4 (PAD4, encoded by Padi4 in mice), an enzyme important in chromatin decondensation, was elevated in neutrophils from individuals with diabetes. When subjected to excisional skin wounds, wild-type (WT) mice produced large quantities of NETs in wounds, but this was not observed in Padi4(-/-) mice. In diabetic mice, higher levels of citrullinated histone H3 (H3Cit, a NET marker) were found in their wounds than in normoglycemic mice and healing was delayed. Wound healing was accelerated in Padi4(-/-) mice as compared to WT mice, and it was not compromised by diabetes. DNase 1, which disrupts NETs, accelerated wound healing in diabetic and normoglycemic WT mice. Thus, NETs impair wound healing, particularly in diabetes, in which neutrophils are more susceptible to NETosis. Inhibiting NETosis or cleaving NETs may improve wound healing and reduce NET-driven chronic inflammation in diabetes.

Deep vein thrombosis and pulmonary embolism are major health problems associated with high mortality. Recently, DNA-based neutrophil extracellular traps (NETs) resulting from the release of decondensed chromatin, were found to be part of the thrombus scaffold and to promote coagulation. However, the significance of nuclear decondensation and NET generation in thrombosis is largely unknown. To address this, we adopted a stenosis model of deep vein thrombosis and analyzed venous thrombi in peptidylarginine deiminase 4 (PAD4)-deficient mice that cannot citrullinate histones, a process required for chromatin decondensation and NET formation. Intriguingly, less than 10% of PAD4 −/− mice produced a thrombus 48 h after inferior vena cava stenosis whereas 90% of wild-type mice did. Neutrophils were abundantly present in thrombi formed in both groups, whereas extracellular citrullinated histones were seen only in thrombi from wild-type mice. Bone marrow chimera experiments indicated that PAD4 in hematopoietic cells was the source of the prothrombotic effect in deep vein thrombosis. Thrombosis could be rescued by infusion of wild-type neutrophils, suggesting that neutrophil PAD4 was important and sufficient. Endothelial activation and platelet aggregation were normal in PAD4 −/− mice, as was hemostatic potential determined by bleeding time and platelet plug formation after venous injury. Our results show that PAD4-mediated chromatin decondensation in the neutrophil is crucial for pathological venous thrombosis and present neutrophil activation and PAD4 as potential drug targets for deep vein thrombosis.

Neutrophil recruitment to the inflammatory sites is regulated by a variety of adhesion molecules including β2 integrins. The dependency of neutrophil recruitment on β2 integrins is variable in different tissues, but has not yet been verified in the cutaneous passive reverse Arthus reaction. We examined this question and also evaluated the impact of isoflurane on neutrophil recruitment to the skin because we previously showed in vitro that isoflurane binds and inhibits β2 integrins. The dependency on β2 integrins in neutrophil recruitment to the skin in the Arthus reaction was examined using αL, αM and β2 knockout mice. Then, we evaluated the effect of isoflurane on neutrophil recruitment to the skin. In addition, the effects of isoflurane on neutrophil binding to intercellular adhesion molecule-1 (ICAM-1), one of the β2 integrin ligands, were studied in vitro using cell adhesion assays. Neutrophil recruitment to the skin in the Arthus reaction model was totally dependent on β2 integrins, as β2 knockout mice completely abolished it. However, the defect of only one of the β2 integrins was not sufficient to abolish neutrophil recruitment. Isoflurane reduced neutrophil recruitment to the skin by approximately 90 %. Also, isoflurane inhibited neutrophil adhesion to β2 integrin ligand ICAM-1. We demonstrated that (1) neutrophil recruitment to the skin was totally dependent on β2 integrins, and (2) isoflurane significantly impaired neutrophil recruitment. Based on the previous studies on the contribution of other adhesion molecules in neutrophil recruitment, it is likely that isoflurane at least partially affects on β2 integrins in this model.

Cancer-associated thrombosis often lacks a clear etiology. However, it is linked to a poor prognosis and represents the second-leading cause of death in cancer patients. Recent studies have shown that chromatin released into blood, through the generation of neutrophil extracellular traps (NETs), is procoagulant and prothrombotic. Using a murine model of chronic myelogenous leukemia, we show that malignant and nonmalignant neutrophils are more prone to NET formation. This increased sensitivity toward NET generation is also observed in mammary and lung carcinoma models, suggesting that cancers, through a systemic effect on the host, can induce an increase in peripheral blood neutrophils, which are predisposed to NET formation. In addition, in the late stages of the breast carcinoma model, NETosis occurs concomitant with the appearance of venous thrombi in the lung. Moreover, simulation of a minor systemic infection in tumor-bearing, but not control, mice results in the release of large quantities of chromatin and a prothrombotic state. The increase in neutrophil count and their priming is mediated by granulocyte colony-stimulating factor (G-CSF), which accumulates in the blood of tumor-bearing mice. The prothrombotic state in cancer can be reproduced by treating mice with G-CSF combined with low-dose LPS and leads to thrombocytopenia and microthrombosis. Taken together, our results identify extracellular chromatin released through NET formation as a cause for cancer-associated thrombosis and unveil a target in the effort to decrease the incidence of thrombosis in cancer patients.

SummaryRecombinant tissue plasminogen activator (r-tPA) is the drug of choice for thrombolysis, but it is associated with a significant risk of bleeding and is not always successful. By cleaving von Willebrand factor (VWF), the metalloprotease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats-13) down-regulates thrombus formation in injured vessels. We investigated whether recombinant ADAMTS13 (r-ADAMTS13) induces thrombolysis in vivo in mice. Thrombosis was produced by ferric chloride-induced (FeCl3) injury in the venules of a dorsal skinfold chamber. Phosphate-buffered saline (PBS, vehicle), r-tPA or r-ADAMTS13, supplemented with hirudin (to stop ongoing thrombin generation), was directly applied onto the occluded vessel, and thrombus dissolution was evaluated by intravital microscopy. The incidence of blood flow restoration significantly increased 30 minutes (min) after r-ADAMTS13 vs. PBS treatment (60% vs. 0%, p

Platelets are crucial regulators of tumor vascular homeostasis and continuously prevent tumor hemorrhage through secretion of their granules. However, the reason for tumor bleeding in the absence of platelets remains unknown. Tumors are associated with inflammation, a cause of hemorrhage in thrombocytopenia. Here, we investigated the role of the inflamed tumor microenvironment in the induction of tumor vessel injury in thrombocytopenic mice. Using s.c. injections of vascular endothelial growth factor or tumor necrosis factor-alpha combined with depletion of neutrophils, we demonstrate that enhancing the opening of endothelial cell junctions was not sufficient to cause bleeding in the absence of platelets; instead, induction of tissue hemorrhage in thrombocytopenia required recruitment of leukocytes. Immunohistology revealed that thrombocytopenia-induced tumor hemorrhage occurs at sites of macrophage and neutrophil accumulation. Mice deficient in beta2 or beta3 integrins, which have decreased neutrophil and/or macrophage infiltration in their tumor stroma, were protected from thrombocytopenia-induced tumor hemorrhage, indicating that, in the absence of platelets, stroma-infiltrating leukocytes induced tumor vessel injury. This injury was independent of reactive oxygen species generation and of complement activation, as suggested by the persistence of tumor hemorrhage in C3- and nicotinamide adenine dinucleotide phosphate oxidase-deficient thrombocytopenic mice. Our results show that platelets counteract tumor-associated inflammation and that the absence of this platelet function elicits vascular injuries by tumor-infiltrating innate immune cells.

Galectin-7 is found mainly in stratified squamous epithelia as well as in various other types of cancer cells. As with other members of the galectin family, the expression of galectin-7 has been shown to negatively regulate the development of some tumors while correlating with the progression of other tumor types. For example, up-regulation of galectin-7 is associated with rat mammary carcinomas and with progression to T-cell malignancy. Here, we provide evidence indicating that galectin-7 functions as an important molecule in the dissemination of lymphoma cells in vivo. We found that stable transfection of lymphoma cells with a plasmid encoding antisense galectin-7 cDNA significantly inhibited the dissemination and invasion of lymphoma cells to peripheral organs, thereby increasing the survival of mice. We also found that inhibition of galectin-7 in aggressive lymphoma cells correlated with a decreased invasion of tumor cells in target organs and a reduced expression of matrix metalloproteinase-9, a gene associated with a poor prognosis in non–Hodgkin's lymphoma. We finally examined the expression of galectin-7 in 50 specimens of different mature B-cell neoplasms and found high galectin-7 expression levels in a significant proportion of mature B-cell neoplasms but not in normal B cells. Taken together, these findings suggest that galectin-7 is a potential therapeutic target in the treatment of lymphoid malignancies. [Cancer Res 2007;67(6):2824–9]

Neutrophil extracellular traps (NETs), consisting of nuclear DNA with histones and microbicidal proteins, are expelled from activated neutrophils during sepsis. NETs were shown to trap microbes, but they also fuel cardiovascular, thrombotic, and autoimmune disease. The role of NETs in sepsis, particularly the balance between their antimicrobial and cytotoxic actions, remains unclear. Neutrophils from peptidylarginine deiminase 4-(PAD4(-/-)) deficient mice, which lack the enzyme allowing for chromatin decondensation and NET formation, were evaluated. We found that neutrophil functions involved in bacterial killing, other than NETosis, remained intact. Therefore, we hypothesized that prevention of NET formation might not have devastating consequences in sepsis. To test this, we subjected the PAD4(-/-) mice to mild and severe polymicrobial sepsis produced by cecal ligation and puncture. Surprisingly, under septic conditions, PAD4(-/-) mice did not fare worse than wild-type mice and had comparable survival. In the presence of antibiotics, PAD4-deficiency resulted in slightly accelerated mortality but bacteremia was unaffected. PAD4(-/-) mice were partially protected from lipopolysaccharide-induced shock, suggesting that PAD4/NETs may contribute to the toxic inflammatory and procoagulant host response to endotoxin. We propose that preventing NET formation by PAD4 inhibition in inflammatory or thrombotic diseases is not likely to increase host vulnerability to bacterial infections.

Genome-wide DNA hypomethylation is a critical mechanism underlying neoplastic transformation. Thus, genes that are suppressed in normal tissues or in specific cell types may become aberrantly expressed in neoplasia. To determine whether DNA methylation can modulate matrix metalloproteinase (mmp) gene expression, we have used a genetically engineered cell line in which both key DNA methyltransferase genes, Dnmt-1 and Dnmt-3b, were removed by homologous recombination. We found that cells bearing a dual knock-out of both Dnmt-1 and Dnmt-3b genes induced de novo expression of mmp-3 gene, but not that of mmp-1 and mmp-2. Furthermore, treatment of the wild-type cells with DNA methylase inhibitors 5-aza-dC and zebularine also induced mmp-3 gene expression. On the other hand, in vitro methylation of the mmp-3 promoter suppressed its transcriptional activity. Finally, we found that induction of mmp-3 and mmp-10 gene expression by hypomethylation was cell-specific, suggesting that epigenetic changes may predispose cells to express stromelysin genes.

We have previously shown that ICAM-1-deficient mice were resistant to lymphoma dissemination of intravenously injected 164T2 lymphoma cells. Highly aggressive variants of this cell line, however, could overcome this resistance. To discern the complex pattern of gene expression involved in the evolution of aggressiveness in lymphoma cells, we compared the transcriptome of 164T2 cells with that of their aggressive variants using cDNA arrays. We identified several genes that were differentially expressed in nonmetastatic lymphoma cells and their metastatic variants. Galectin-7, associated with the development of chemically induced mammary carcinoma, was one such gene whose expression was significantly upregulated. We showed that it was constitutively expressed in aggressive variants, at both mRNA and protein levels. Galectin-7 expression in aggressive lymphoma cells was induced upon in vivo selection in several organs, including the thymus, the spleen and kidneys. We also showed that treatment of nonaggressive lymphoma cells with 5-aza-2'-deoxycytidine was sufficient to induce galectin-7 gene expression. This report is the first to show that galectin-7 is expressed in aggressive lymphoma.

Introduction Histone hypercitrullination by the enzyme peptidylarginine deiminase 4 (PAD4) leads to nuclear chromatin decondensation that is needed for neutrophil extracellular trap (NET) formation. NETs consist of chromatin and granule proteins that are released into the extracellular environment. NETs were shown to be involved in thrombosis by promoting coagulation and platelet adhesion and were identified in the thrombus scaffold in animal models of deep vein thrombosis (DVT). Objective Whether NETs are involved in the pathogenesis of DVT or whether they are merely a consequence of neutrophil recruitment to the thrombus is unknown. We hypothesized that NET formation would be impaired in PAD4-deficient mice during deep vein thrombosis and that this may affect thrombus formation and/or stability. Methods PAD4-deficient mice are incapable of citrullinating histones and therefore fail to decondense chromatin during NETosis. We performed the inferior vena cava stenosis model of DVT in wild-type or PAD4-/- mice. Intravital microscopy was done to assess leukocyte vessel wall interaction in PAD4 deficiency. Results We induced NET formation in isolated peripheral blood mouse neutrophils with ionomycin and found that PAD4-/- neutrophils had a complete inability to produce NETs (WT, 20.65±2.61% NETs; PAD4-/-, not detected. n=4). Leukocyte-endothelial interactions in PAD4-/- mice were not impaired upon induction of systemic Weibel-Palade body release (WT, 55.2±11.8; PAD4-/-, 62.0±17.5 cells/min, n=5-6). In the DVT model, while a majority (9/10) of wild-type mice formed a thrombus 48 hours after stenosis, only 1 of 11 PAD4-/- mice formed a thrombus. Thrombus formation could be rescued by infusions of isolated WT bone marrow neutrophils into PAD4-/- mice, and extracellular H3Cit+ areas were seen within these thrombi. This data suggests that neutrophil PAD4 was essential for thrombus formation in deep veins. Conclusion NETs comprise a crucial part of the pathologic thrombus scaffold, and here we report that the lack of NETs inhibits pathological thrombosis. Chromatin decondensation initiated by PAD4 in neutrophils is a key player in the formation of deep vein thrombi and targeting neutrophil histone modification could be a new way to prevent DVT.

The majority of peripheral serotonin is stored in platelets, which secrete it on activation. Serotonin releases Weibel-Palade bodies (WPBs) and we asked whether absence of platelet serotonin affects neutrophil recruitment in inflammatory responses. Tryptophan hydroxylase (Tph)1–deficient mice, lacking non-neuronal serotonin, showed mild leukocytosis compared with wild-type (WT), primarily driven by an elevated neutrophil count. Despite this, 50% fewer leukocytes rolled on unstimulated mesenteric venous endothelium of Tph1(-/-) mice. The velocity of rolling leukocytes was higher in Tph1(-/-) mice, indicating fewer selectin-mediated interactions with endothelium. Stimulation of endothelium with histamine, a secretagogue of WPBs, or injection of serotonin normalized the rolling in Tph1(-/-) mice. Diminished rolling in Tph1(-/-) mice resulted in reduced firm adhesion of leukocytes after lipopolysaccharide treatment. Blocking platelet serotonin uptake with fluoxetine in WT mice reduced serum serotonin by80% and similarly reduced leukocyte rolling and adhesion. Four hours after inflammatory stimulation, neutrophil extravasation into lung, peritoneum, and skin wounds was reduced in Tph1(-/-) mice, whereas in vitro neutrophil chemotaxis was independent of serotonin. Survival of lipopolysaccharide-induced endotoxic shock was improved in Tph1(-/-) mice. In conclusion, platelet serotonin promotes the recruitment of neutrophils in acute inflammation, supporting an important role for platelet serotonin in innate immunity.

Neutrophils play a major role in cancer biology and both pro- and antitumoral functions of tumor-infiltrating neutrophils have been described. We have shown that tumors, by releasing G-CSF into the bloodstream, prime circulating neutrophils to form neutrophil extracellular traps (NETs) and we have detected the presence of NETs within the tumor microenvironment. Here, we report, using PAD4-deficient mice with a defect in neutrophil chromatin decondensation and NET formation, that the priming of neutrophils toward NETosis favors tumor growth. Interestingly, in a tumor model that does not release G-CSF and in which neutrophils are not primed for NETosis, PAD4-deficiency did not reduce tumor growth. However, supplying exogenous G-CSF to the wild-type (WT) host promoted intratumoral NETosis and tumor growth. Taken together, our results suggest that the priming of neutrophils for NETosis by the tumor or its environment leads to the accumulation of intratumoral NETs and a growth advantage to the tumor. Our work unveiled a pro-tumoral role for NETs which strengthens their potential as a new target in the fight against cancer.

Thrombocytopenia-induced tumor hemorrhage improves drug delivery to tumors. This phenomenon presents a new way to increase drug efficacy with minimal side effects. Combining anti-platelet treatment with therapeutic drugs may help us in the search for more effective ways to fight cancer.

The haemostatic role of platelets was established in the 1880s by Bizzozero who observed their ability to adhere and aggregate at sites of vascular injury. It was only some 80 years later that the function of platelets in maintaining the structural integrity of intact blood vessels was reported by Danielli. Danielli noted that platelets help preserve the barrier function of endothelium during organ perfusion. Subsequent studies have demonstrated further that platelets are continuously needed to support intact mature blood vessels. More recently, platelets were shown to safeguard developing vessels, lymphatics, as well as the microvasculature at sites of leukocyte infiltration, including inflamed organs and tumours. Interestingly, from a mechanistic point of view, the supporting role of platelets in these various vessels does not necessarily involve the well-understood process of platelet plug formation but, rather, may rely on secretion of the various platelet granules and their many active components. The present review focuses on these nonconventional aspects of platelet biology and function by presenting situations in which platelets intervene to maintain vascular integrity and discusses possible mechanisms of their actions. We propose that modulating these newly described platelet functions may help treat haemorrhage as well as treat cancer by increasing the efficacy of drug delivery to tumours.

Platelets contribute to homeostasis of the tumor vasculature by helping prevent hemorrhage. Thus, we hypothesized that inducing thrombocytopenia would increase tumor vascular leakiness and facilitate the effective delivery of chemotherapeutic agents to tumors. In a mammary carcinoma murine model, platelet depletion induced bleeding specifically at the tumor site, favoring the accumulation of fluorescently labeled microspheres only in the tumor. Moreover, induction of thrombocytopenia in tumor-bearing mice before injection of paclitaxel increased its intratumoral accumulation and reduced growth of both slow- and fast-growing tumors, compared with mice with normal platelet counts that were treated only with paclitaxel. Histologic analysis confirmed the expectation of an increase in tumor apoptosis and a reduction in tumor proliferation in thrombocytopenic mice receiving chemotherapy. No increased toxicity was seen in other organs or blood cells. Taken together, our results indicate that low platelet count selectively induces leakiness of tumor vessels and favors the delivery of chemotherapy to tumor sites, enhancing its tumoricidal effects. Cancer Res; 71(5); 1540–9. ©2011 AACR.

International audience; Galectins are members of a family of beta-galactosides-binding proteins that have recently emerged as novel modulators in different aspects of cancer. The expression of galectins in tumors and/or the tissue surrounding them has been well documented. Since galectin-7 expression has been associated with epithelial tissues and varies significantly in various types of cancer, we have investigated for the first time its role in breast cancer. Using two preclinical mouse models, high levels of galectin-7 expression in breast cancer cells drastically increased their ability to metastasize to lungs and bones. Significant increases in the number of pulmonary metastases and osteolytic lesions were induced by overexpression of galectin-7 compared with control cells. In human tissues, galectin-7 was specifically found in myoepithelial cells of normal human breast tissue, but not in luminal cells. Its expression was severely altered in breast carcinoma, many samples showing greater than 70% of galectin-7 positive cells. High expression levels of galectin-7 were restricted to high-grade breast carcinomas, including HER2 overexpressing and basal-like groups. In HER2 overexpressing cases, galectin-7 expression was associated with lymph node axillary metastasis. Taken together, our results indicate that galectin-7 may represent a potential target for both specific detection and therapeutic inhibition of metastatic breast cancer.

Recent studies have reported that elevated levels of galectin-7 in different types of cancer. The mechanisms underlying its abnormal regulation in cancer cells remain, however, unknown. Here, we have examined the relationship between galectin-7 and p53, a gene previously associated with upregulation of galectin-7. While RNA and protein analyses revealed a consistent and irreversible upregulation of galectin-7 throughout progression of lymphoma, no correlation with p53 was found. In fact, most of the lymphoma cell lines expressing high levels of galectin-7 did not express any detectable level of p53, although expressed p21(Waf1/Cip1) gene following doxorubicin treatment, indicating that p53 was functional in these cells. Methylation-specific polymerase chain reaction (MS-PCR) analyses rather suggested that galectin-7 expression was associated with changes in DNA methylation. This conclusion was supported by data using demethylating agent 5-aza-dC. Furthermore, disruption of the DNA methylases dnmt1 and dnmt3a induced galectin-7. Collectively, our data suggest that abnormal expression of galectin-7 in lymphoma cells is not dependent on p53, but is rather associated with DNA hypomethylation.

Previous studies have shown that high levels of MMP-9 can be detected in the serum of patients with various lymphoid malignancies and in leukemia/lymphoma culture supernatants. Indeed, aggressive forms of lymphoma constitutively produce MMP-9 and its elevated levels in the serum or in tissues correlate with advanced stage and poor patient survival. In vitro, MMP-9, which is also produced by the host peritumoral cells in response to the presence of tumors, plays an important role in migration of tumor cells through artificial basement membranes or endothelial cells. In this study, using MMP-9-deficient mice, we show that absence of MMP-9 does not prevent the development of primary T-cell leukemia. Furthermore, MMP-9-deficient cell lines retained their tumorigenic potential, as shown by their ability to induce thymic lymphoma in young syngeneic wild-type animals. In addition, these MMP-9-deficient tumor cells disseminate in normal mice, or mice that are deficient for MMP-9, indicating that tumor growth and dissemination can occur in total absence of MMP-9. These results show for the first time than lymphoma growth can occur in total absence of MMP-9 and have consequences for therapy of invasive cancers with inhibitors of MMPs.

Proteins of the Bin-amphiphysin-Rvs (BAR) and Fes-CIP4 homology BAR (F-BAR) families bind and deform membranes, promoting in cells tubular invaginations reminiscent of the platelet open canalicular system (OCS) and megakaryocyte (MK) demarcation membrane system (DMS). Here we investigated the role of the F-BAR protein PACSIN2 in platelets and MKs. Spinning disk laser fluorescence confocal microscopy showed that PACSIN2 co-localized with the von Willebrand factor receptor subunit GPIbα at the openings of the OCS in platelets. Endogenous and over-expressed PACSIN2 associated with anastomosing tubular structures reminiscent of the pre-DMS in mouse bone marrow MKs. Pacsin2-null mice had mild thrombocytopenia and polycythemia, MK hyperplasia and splenomegaly. Pacsin2-null mice had a bleeding disorder, as evidenced by significantly prolonged tail bleeding and delayed in vivo thrombus formation following FeCl3 vascular injury, which was normalized by injection of WT platelets. However, Pacsin2-null platelets expressed P-selectin and activated the integrin αIIbβ3 normally in response to stimulation with thrombin and the GPVI-specific collagen-related peptide. In contrast, Pacsin2-null platelets had abnormal flattened morphology, with more and narrower OCS channels on their surface, compared to WT platelets. Together, the data indicate that the F-BAR protein PACSIN2 orchestrates the intracellular membrane architecture of platelets, thereby regulating their in vivo hemostatic functions. Disclosures No relevant conflicts of interest to declare.

Metastasis is a multistep process by which cancer cells, after acquiring several capabilities, spread to distinct sites in the body. It is the major cause of death in individuals suffering from cancer. We have recently identified galectin-7 as a new gene associated with the progression of T cell lymphoma toward a metastatic phenotype, suggesting a possible causal relationship. The present study was designed to investigate the role of galectin-7 in lymphoma. We found that the development of thymic lymphoma was accelerated when induced by lymphoma cells overexpressing galectin-7. Moreover, transfection of an expression vector containing the galectin-7 gene in low metastatic lymphoma cells increased their metastatic behavior and confers these cells with the new ability to overcome the resistance of intercellular adhesion molecule-1–deficient mice to lymphoma dissemination. Finally, we provide data suggesting that galectin-7 modulates the aggressive behavior of lymphoma cells by controlling the expression of metastatic genes, such as MMP-9. This hypothesis is based on the following evidence: (a) galectin-7 transfectants have higher levels of MMP-9 expression, (b) addition of β-lactose completely inhibits expression of MMP-9 by galectin-7 transfectants, and (c) recombinant forms of galectin-7 induces the expression of MMP-9 in both mouse and human lymphoma cells. Our results have uncovered the existence of a previously undescribed activity, the promotion of cancer cell malignancy, to galectin-7.

Cancers prime neutrophils to release extracellular DNA traps through the systemic release of granulocyte colony-stimulating factor (G-CSF). We recently showed that these circulating neutrophil extracellular traps (NETs) promote the establishment of a pro-thrombotic state. The role of NETs in cancer biology and tumor progression may prove much more than an unfortunate side effect of cancer.

Abstract 2105 Important research goals in cancer therapy include delivering high doses of drug to tumor sites for maximum treatment efficacy while minimizing side effects to healthy organs. We have recently demonstrated that platelets contribute to tumor vascular homeostasis through prevention of severe tumor hemorrhage. In order to define a new way of increasing drug delivery to the tumor site, we hypothesized that the selective induction of tumor vascular leakiness by thrombocytopenia may facilitate the delivery of chemotherapeutic agents to tumors. Using a mammary carcinoma murine model, we showed that platelet depletion induced hemorrhage specifically at the tumor site and favored the accumulation of fluorescently-labeled microspheres in the tumor. Moreover, the induction of thrombocytopenia in tumor-bearing mice before injection of the chemotherapeutic drug paclitaxel significantly reduced tumor growth compared to mice with normal platelet count that were treated with paclitaxel. Analysis of the tumors ex vivo revealed an increase in tumor apoptosis and a reduction in tumor cell proliferation in thrombocytopenic mice that received therapy. No increased toxicity was seen on other organs and blood cells. Taken together, our results indicate that low platelet count selectively induces leakiness of tumor vessels and allows better access of a chemotherapeutic agent to the tumor, enhancing its tumoricidal effect. Disclosures: No relevant conflicts of interest to declare.

In a recent study, we showed that platelets are crucial regulators of tumor vascular homeostasis in that they continuously prevent tumor hemorrhage through secretion of their granules. However, it remains unclear what platelets target to prevent tumor bleeding. Tumors are associated with inflammation, a cause of hemorrhage in thrombocytopenia. We hypothesized that platelets protect tumors from vascular damages induced by infiltrating inflammatory cells. Here, we report that thrombocytopenia-induced tumor hemorrhage preferentially occurs at the periphery of tumors, where massive accumulation of monocytes/macrophages and neutrophils was revealed by tumor histology. To further investigate the role of tumor-infiltrating leukocytes in the induction of tumor hemorrhage in thrombocytopenic mice, we used genetically-engineered mice with a deficiency in either beta2 or beta3 integrins, or in integrin activation. We show that these mice have decreased leukocyte infiltration in the tumor stroma and are protected from thrombocytopenia-induced tumor hemorrhage. Using a model of TNFa-induced skin inflammation, we demonstrate that releasates from collagen- and thrombin-stimulated platelets inhibit neutrophil-induced vascular damage. Our results show that platelets protect tumors from vascular leakage induced by infiltrating leukocytes and that releasate from activated platelets has potent in vivo healing properties needed after leukocyte transmigration.

To form tumors successfully at sites remote from the primary tumor, metastatic cells must be endowed with particular properties. They must detach from the primary tumor and enter the blood circulation, where they must resist hemodynamic shearstress, "home" to the target organ, successfully extravasate, and then migrate through dense stroma to a site favorable for tumor growth. Recent results with genetically engineered mouse models have generated data which clearly challenge the classic dogma stating that matrix metalloproteinases (MMPs) promote metastasis solely by modulating the remodeling of extracellular matrix (ECM). Instead, it is becoming clear that MMPs and their natural inhibitors have multiple biological functions that not only challenge our view on how MMPs promote metastasis, but also raise for the first time the idea that secretion of MMPs by the host could protect it from tumor growth, at least in some types of cancer or at specific stages of tumor progression.