Anticoagulant use in patients with cancer associated venous thromboembolism: a retrospective cohort study.

INTRODUCTION: Long term anticoagulant therapy is recommended for treatment and secondary prevention of venous thromboembolism in cancer patients. We assessed outpatient anticoagulants [warfarin, low molecular weight heparins (LMWHs), fondaparinux and unfractionated heparin (UFH)] use in adult, cancer patients, 20years of age or older, who incurred a venous thromboembolism (primary or secondary in-hospital diagnosis) in Quebec, Canada between 2007 and 2009. MATERIALS AND METHODS: Data were obtained from the Quebec Health Insurance Agency. Patients with an in-hospital cancer diagnosis between April 2007 and June 2009 and an in-hospital venous thromboembolism diagnosis either concurrently or consequently were eligible at the date of discharge (index date). Those patients registered with the provincial drug plan and discharged to the community were included in the study and followed for 6months. RESULTS: Among 2,070 study patients, 72.4% received anticoagulant therapy at index date, 60% of whom were persistent with therapy and received it for ≥80% of follow-up days. Outpatient anticoagulant use was more likely in those with primary versus secondary diagnosis of venous thromboembolism and less likely in patients with cerebrovascular disease, peptic ulcer disease or previous anticoagulant use. The small number of patients who used either UFH (n=11) or fondaparinux (n=5) at index date were included in the LMWH group. Warfarin use was less likely than LMWH use in corticosteroid users, previous anticoagulant users, patients with metastatic cancer and those with catheter or chemotherapy in the previous three months. Warfarin use was more likely than LMWH use in: older patients, those residing in rural areas, those with lower income and those suffering from ischemic heart disease, atrial fibrillation or chronic kidney disease. Patients with ischemic heart disease were more likely to have used a non-dalteparin LMWH versus dalteparin (currently, the only LMWH approved by health Canada for chronic treatment of VTE), while those residing in rural areas and those with catheter/chemotherapy were less likely to have used them. A primary (versus secondary) discharge diagnosis of venous thromboembolism [Odds Ratio 1.42; 95% confidence interval (1.14, 1.76)], and metastatic cancer 1.27 (1.00, 1.60) were associated with persistence on anticoagulant treatment. CONCLUSION: Guideline recommended outpatient use of anticoagulant in cancer patients hospitalized with venous thromboembolism was influenced by cancer status, old age and low income. Risk factors for bleeding prevented outpatient anticoagulant use in some patients.

Genetic and pharmacological evaluation of cathepsin s in a mouse model of asthma.

Cathepsin S (Cat S) is predominantly expressed in antigen-presenting cells and is up-regulated in several preclinical models of antigen-induced inflammation, suggesting a role in the allergic response. Prophylactic dosing of an irreversible Cat S inhibitor has been shown to attenuate pulmonary eosinophilia in mice, supporting the hypothesis that Cat S inhibition before the initiation of airway inflammation is beneficial in airway disease. In addition, Cat S has been shown to play a role in more distal events in the allergic response. To determine where Cat S inhibition may affect the allergic response, we used complementary genetic and pharmacological approaches to investigate the role of Cat S in the early and downstream allergic events in a murine model of antigen-induced lung inflammation. Cat S knockout mice did not develop ovalbumin-induced pulmonary inflammation, consistent with a role for Cat S in the development of the allergic response. Alternatively, wild-type mice were treated with a reversible, highly selective Cat S inhibitor in prophylactic and therapeutic dosing paradigms and assessed for changes in airway inflammation. Although both treatment paradigms resulted in potent Cat S inhibition, only prophylactic Cat S inhibitor dosing blocked lung inflammation, consistent with our findings in Cat S knockout mice. The findings indicate that although Cat S is up-regulated in allergic models, it does not appear to play a significant role in the downstream effector inflammatory phase in this model; however, our results demonstrate that Cat S inhibition in a prophylactic paradigm would ameliorate airway inflammation.

Adenovirus IL-13-induced airway disease in mice: a corticosteroid-resistant model of severe asthma.

Interleukin 13 (IL-13) is considered to be a key driver of the development of airway allergic inflammation and remodeling leading to airway hyperresponsiveness (AHR). How precisely IL-13 leads to the development of airway inflammation, AHR, and mucus production is not fully understood. In order to identify key mediators downstream of IL-13, we administered adenovirus IL-13 to specifically induce IL-13-dependent inflammation in the lungs of mice. This approach was shown to induce cardinal features of lung disease, specifically airway inflammation, elevated cytokines, AHR, and mucus secretion. Notably, the model is resistant to corticosteroid treatment and is characterized by marked neutrophilia, two hallmarks of more severe forms of asthma. To identify IL-13-dependent mediators, we performed a limited-scale two-dimensional SDS-PAGE proteomic analysis and identified proteins significantly modulated in this model. Intriguingly, several identified proteins were unique to this model, whereas others correlated with those modulated in a mouse ovalbumin-induced pulmonary inflammation model. We corroborated this approach by illustrating that proteomic analysis can identify known pathways/mediators downstream of IL-13. Thus, we have characterized a murine adenovirus IL-13 lung model that recapitulates specific disease traits observed in human asthma, and have exploited this model to identify effectors downstream of IL-13. Collectively, these findings will enable a broader appreciation of IL-13 and its impact on disease pathways in the lung.

Pulmonary oxidant stress in murine sepsis is due to inflammatory cell nitric oxide.

OBJECTIVE: Pulmonary oxidant stress is an important pathophysiologic feature of acute lung injury. It is unclear whether nitric oxide contributes to this oxidant stress. Thus, we examined the role of inducible nitric oxide synthase (iNOS) in pulmonary oxidant stress in murine sepsis and the differential contribution of different cellular sources of iNOS. DESIGN: Randomized, controlled animal study. SETTING: Research laboratory of an academic institution. SUBJECTS: Male iNOS+/+, iNOS-/- C57Bl/6 mice, and bone-marrow transplanted iNOS chimeric mice: +to- (wild-type iNOS+/+ donor bone-marrow transplanted into iNOS-/- recipient mice) and the reciprocal -to+ chimeras. INTERVENTIONS: Animals were randomized to sepsis (n = 264), induced by cecal ligation and perforation, vs. naive groups (n = 138). MEASUREMENTS AND MAIN RESULTS: In septic iNOS-/- vs. wild-type iNOS+/+ mice, sepsis-induced pulmonary oxidant stress (33 +/- 11 [mean +/- sem] vs. 365 +/- 48 pg 8-isoprostane/mg protein, p < .01) and nitrosative stress (0.0 +/- 0.0 vs. 0.9 +/- 0.4 micromol 3-nitrotyrosine/mmol para-tyrosine, p < .05) were abolished, despite similar septic increases in pulmonary myeloperoxidase activity in both (86 +/- 20 vs. 83 +/- 12 mU/mg protein, p = .78). In +to- iNOS chimeric mice (iNOS localized only to donor bone-marrow-derived inflammatory cells), cecal ligation and perforation resulted in significant pulmonary oxidant stress (368 +/- 81 pg 8-isoprostane/mg protein) and nitrosative stress (0.6 +/- 0.2 micromol 3-nitrotyrosine/mmol para-tyrosine), similar in degree to septic wild-type mice. In contrast, pulmonary oxidant and nitrosative stresses were absent in septic -to+ iNOS chimeras (iNOS localized only to recipient parenchymal cells), similar to iNOS-/- mice. CONCLUSIONS: In murine sepsis-induced acute lung injury, pulmonary oxidant stress is completely iNOS dependent and is associated with tyrosine nitration. Moreover, pulmonary oxidant stress and nitrosative stress were uniquely dependent on the presence of iNOS in inflammatory cells (e.g., macrophages and neutrophils), with no apparent contribution of iNOS in pulmonary parenchymal cells. iNOS inhibition targeted specifically to inflammatory cells may be an effective therapeutic approach in sepsis and acute lung injury.

Pulmonary neutrophil infiltration in murine sepsis: role of inducible nitric oxide synthase.

Nitric oxide (NO) derived from inducible NO synthase (iNOS) contributes to the pathophysiology of acute lung injury (ALI). The effect of iNOS on pulmonary neutrophil infiltration in ALI is not known. Thus, we assessed pulmonary microvascular neutrophil sequestration through intravital videomicroscopy and pulmonary neutrophil infiltration, reflected by myeloperoxidase activity and lavage neutrophil counts, after induction of sepsis by cecal ligation/perforation in wild-type (iNOS+/+) versus iNOS-/- mice. Pulmonary microvascular neutrophil sequestration was attenuated in septic iNOS-/- versus iNOS+/+ mice (15 +/- 1 vs. 20 +/- 1 leukocytes per field, p < 0.05), but lavage neutrophil counts were greater in iNOS-/- mice (5.7 +/- 1.5% vs. 0.7 +/- 0.1%, p < 0.05) between 6 and 18 hours after cecal ligation and perforation. When iNOS+/+ bone marrow was transplanted into bone marrow-depleted iNOS-/- mice (+ to - chimeras; iNOS limited to marrow-derived inflammatory cells), septic pulmonary microvascular neutrophil sequestration and lavage neutrophil counts were restored to levels seen in septic iNOS+/+ mice. In contrast, in - to + chimeras, pulmonary neutrophil trafficking was similar to iNOS-/- mice. In vitro cytokine-stimulated neutrophil transendothelial migration was significantly greater for iNOS-/- versus iNOS+/+ neutrophils (7.9 +/- 0.7% vs. 3.8 +/- 0.6%, p < 0.05) but was independent of endothelial iNOS. Thus, neutrophil iNOS-derived NO is an important autocrine modulator of pulmonary neutrophil infiltration in murine sepsis.

Excess nitric oxide decreases cytochrome P-450 2J4 content and P-450-dependent arachidonic acid metabolism in lungs of rats with acute pneumonia.

Recently, we demonstrated that pulmonary CYP2J4 content, a prominent source of EETs and HETEs formation in rat lungs, is reduced in pneumonia. Therefore, the purpose of this study was to determine the role of iNOS-derived NO in reduced pulmonary CYP2J4 protein content and decreased CYP metabolites in pneumonia. Rats were randomized to control, control plus 1400W (iNOS inhibitor), pneumonia, and pneumonia plus 1400W groups. Pseudomonas organisms were injected into lungs of pneumonia rats. At 40 h after surgery, rats were treated with either saline or 1400W for 4 h before death. Venous plasma samples were obtained for measuring nitrites/nitrates (NOx). There was no significant effect of 1400W on blood pressure measured in control or pneumonia rats, whereas 1400W reduced the elevated plasma NOx levels in pneumonia rats by half. CYP primary metabolites of AA formed at significantly lower rates in pulmonary microsomes from pneumonia rats compared with control rats. Treatment of pneumonia rats with 1400W resulted in a significant increase in the rate of formation of pulmonary EETs and omega-terminal HETEs compared with untreated pneumonia rats. The reduction in CYP2J4 protein content in pneumonia lung microsomes was also partially prevented by 1400W. Therefore, excess NO from iNOS decreases the pulmonary production of EETs and omega-HETEs in acute pneumonia. Inhibition of iNOS restores CYP2J4 protein content and CYP activity in acute pneumonia, indicating an important NO-CYP interaction in pulmonary responses to infection. We speculate CYP2J4 and its AA metabolites are involved in the modulation of pulmonary function in health and disease.

Effects of nebulized diethylenetetraamine-NONOate in a mouse model of acute Pseudomonas aeruginosa pneumonia.

OBJECTIVE: Endogenous and exogenous nitric oxide (NO) may have important antibacterial effects in patients with pneumonia. NO administration has been limited to the continuous inhalation of gas-phase NO (ie, inhaled NO [iNO]). Intermittent nebulization of NONOates, novel NO donors, may permit the continuous intrapulmonary delivery of NO. Thus, we assessed the effects of nebulized diethylenetetraamine-NONOate (DETA-NO) in a model of acute Pseudomonas aeruginosa pneumonia. DESIGN: Randomized, controlled study. SUBJECTS: Male C57Bl/6 mice. INTERVENTIONS: Pneumonia was induced by intratracheal instillation of P aeruginosa (3 x 10(7) CFU in 50 microL). Pneumonia and sham mice were randomized to receive no treatment, nebulized DETA-NO (12.5 or 125 micromol) at 4 h and 12 h, or continuous iNO for 24 h (10 or 40 ppm) until they were killed at 24 h. MAIN RESULTS: The nebulization of DETA-NO was associated with a marked increase in mean (+/- SEM) exhaled NO levels (after nebulization, 484 +/- 34 parts per billion [ppb]; baseline, 13.4 +/- 0.4 ppb; p < 0.01) and plasma levels of nitrites/nitrates (after nebulization, 73 +/- 28 microM; at baseline, 14 +/- 3 microM; p < 0.05). Nebulized DETA-NO decreased the pulmonary bacterial load in mice with pneumonia by 65 +/- 19% (p < 0.05 vs untreated mice) but had no effect on pulmonary leukocyte infiltration. Although the growth of P aeruginosa colonies in vitro was impaired on exposure to DETA-NO, growth was similarly impaired by exposure to DETA nucleophile/backbone alone. CONCLUSIONS: The nebulization of DETA-NO provides a method for the prolonged intrapulmonary delivery of NO. The antibacterial effect of DETA-NO in vivo and in vitro is due, in large part, to the DETA nucleophile moiety and is independent of NO, suggesting a limited therapeutic role for exogenous NO in pneumonia.

Role of inducible nitric oxide synthase in pulmonary microvascular protein leak in murine sepsis.

The effects of nitric oxide (NO) from calcium-independent NO synthase (iNOS) on microvascular protein leak in acute lung injury (ALI) are uncertain, possibly because of disparate effects of iNOS-derived NO from different cells. We assessed the contribution of iNOS from inflammatory versus parenchymal cells to pulmonary protein leak in murine cecal ligation and perforation-induced ALI. We studied iNOS+/+, iNOS-/-, and two reciprocally bone marrow-transplanted iNOS chimeric mice groups: + to - (iNOS+/+ donor bone marrow-transplanted into iNOS-/- recipient mice) and - to +. Sepsis-induced ALI was characterized by pulmonary leukocyte infiltration, increased pulmonary iNOS activity, and increased pulmonary microvascular protein leak, as assessed by Evans blue (EB) dye. Despite equal neutrophil infiltration, sepsis-induced EB-protein leak was eliminated in iNOS-/- mice and in - to + iNOS chimeras (parenchymal cell-localized iNOS) but was preserved in + to - chimeric mice (inflammatory cell-localized iNOS). EB-protein leak was also prevented by pretreatment with allopurinol and superoxide dismutase. Microvascular protein leak in sepsis-induced ALI is uniquely dependent on iNOS in inflammatory cells with no obvious contribution of iNOS in pulmonary parenchymal cells. Pulmonary protein leak is also dependent on superoxide, suggesting an effect of peroxynitrite rather than NO itself.

Effects of inhaled nitric oxide in a mouse model of sepsis-induced acute lung injury.

OBJECTIVE: Although inhaled nitric oxide transiently improves oxygenation in patients with acute lung injury, it has not affected clinical outcomes. As well, the effects of inhaled nitric oxide on the pathophysiologic features of acute lung injury have not been well defined. Therefore, we assessed the effects of inhaled nitric oxide on the degree of pulmonary inflammation and injury in a mouse model of sepsis-induced acute lung injury. DESIGN: Randomized, controlled animal study. SETTING: Research laboratory of an academic institution. SUBJECTS: Male C57Bl/6 mice. INTERVENTIONS: Sepsis was induced by cecal ligation and perforation. At the time of surgery, septic and naïve mice were randomized to exposure to either 40 ppm inhaled nitric oxide or room air for 24 hrs before they were killed. MEASUREMENTS AND MAIN RESULTS: Sepsis-induced acute lung injury was characterized by increased pulmonary myeloperoxidase (68 +/- 13 vs. 13 +/- 3 mU/mg protein in naïve mice, p <.01), pulmonary 8-isoprostane content (627 +/- 51 vs. 88 +/- 20 pg/mg protein in naïve mice, p <.01), and protein in bronchoalveolar lavage fluid (p <.05). Inhaled nitric oxide exposure in septic mice completely abrogated the septic increases in myeloperoxidase activity (p <.05) and pulmonary 8-isoprostane content (p <.05) but had no effect on bronchoalveolar lavage protein. The induction of sepsis also was associated with an increase in pulmonary inducible NO synthase activity (2.8 +/- 0.5 vs. 0.4 +/- 0.1 pmol small middle dotmin-1 small middle dotmg-1 protein in naïve mice, p <.05), and inhaled nitric oxide attenuated this increase in pulmonary inducible NO synthase activity (p <.05). CONCLUSIONS: Exposure to inhaled nitric oxide early in the course of sepsis-induced acute lung injury is associated with reduced pulmonary leukocyte infiltration and less oxidative injury. Decreased lung inflammation and injury with inhaled nitric oxide is associated with decreased pulmonary inducible NO synthase activity. Therefore, inhaled NO may have greater clinical benefit if administered earlier in the natural history of acute lung injury in patients.