Secreted phospholipase A2 group X overexpression in asthma and bronchial hyperresponsiveness.

RATIONALE: Secreted phospholipase A(2) enzymes (sPLA(2)s) play key regulatory roles in the biosynthesis of eicosanoids, such as the cysteinyl leukotrienes, but the role of these enzymes in the pathogenesis of asthma is not known. OBJECTIVES: To establish if sPLA(2)s are overexpressed in the airways of patients with asthma, and to determine if these enzymes may play a role in the generation of eicosanoids in exercise-induced bronchoconstriction. METHODS: Induced sputum samples were obtained from subjects with asthma with exercise-induced bronchoconstriction and nonasthmatic control subjects at baseline, and on a separate day 30 minutes after exercise challenge. The expression of the PLA(2)s in induced sputum cells and supernatant was determined by quantitative polymerase chain reaction, immunocytochemistry, and Western blot. MEASUREMENTS AND MAIN RESULTS: The sPLA(2)s expressed at the highest levels in airway cells of subjects with asthma were groups X and XIIA. Group X sPLA(2) (sPLA(2)-X) was differentially overexpressed in asthma and localized to airway epithelial cells and bronchial macrophages. The gene expression, immunostaining in airway epithelial cells and bronchial macrophages, and the level of the extracellular sPLA(2)-X protein in the airways increased in response to exercise challenge in the asthma group, whereas the levels were lower and unchanged after challenge in nonasthmatic control subjects. CONCLUSIONS: Increased expression of sPLA(2)-X may play a key role in the dysregulated eicosanoid synthesis in asthma.

Moderate hypothermia (30 degrees C) maintains myocardial integrity and modifies response of cell survival proteins after reperfusion.

Hypothermia preserves myocardial function, promotes signaling for cell survival, and inhibits apoptotic pathways during 45-min reperfusion. We tested the hypothesis that signaling at the transcriptional level is followed by corresponding proteomic response and maintenance of structural integrity after 3-h reperfusion. Isolated hearts were Langendorff perfused and exposed to mild (I group; n = 6, 34 degrees C) or moderate (H group; n = 6, 30 degrees C) hypothermia during 120-min total ischemia with cardioplegic arrest and 180-min 37 degrees C reperfusion. Moderate hypothermia suppressed anaerobic metabolism during ischemia and significantly diminished left ventricular end-diastolic pressure at the end of ischemia from 52.7 +/- 3.3 (I group) to 1.8 +/- 0.9 (H group) mmHg. Unlike the I group, which showed poor cardiac function and high left ventricular pressure, the H group showed preservation of myocardial function, coronary flow, and oxygen consumption. Compared with normal control hearts without ischemia (n = 5), histological staining in the I group showed marked disarray and fragmentation of collagen network (score 4-5), while the H group showed preserved collagen integrity (score 0-1). The apoptosis-linked tumor suppressor protein p53 was expressed throughout the I group only (score 4-5). The H group produced elevated expression for hypoxia-inducible factor 1alpha and heme oxygenase 1, but minimally affected vascular endothelial growth factor expression. The H group also elevated expression for survival proteins peroxisomal proliferator-activated receptor-beta and Akt-1. These results show in a constant left ventricular volume model that moderate hypothermia (30 degrees C) decreases myocardial energy utilization during ischemia and subsequently promotes expression of proteins involved in cell survival, while inhibiting induction of p53 protein. These data also show that 34 degrees C proffers less protection and loss of myocardial integrity.

Importance of group X-secreted phospholipase A2 in allergen-induced airway inflammation and remodeling in a mouse asthma model.

Arachidonic acid metabolites, the eicosanoids, are key mediators of allergen-induced airway inflammation and remodeling in asthma. The availability of free arachidonate in cells for subsequent eicosanoid biosynthesis is controlled by phospholipase A(2)s (PLA(2)s), most notably cytosolic PLA(2)-alpha. 10 secreted PLA(2)s (sPLA(2)s) have also been identified, but their function in eicosanoid generation is poorly understood. We investigated the role of group X sPLA(2) (sPLA(2)-X), the sPLA(2) with the highest in vitro cellular phospholipolysis activity, in acute and chronic mouse asthma models in vivo. The lungs of sPLA(2)-X(-/-) mice, compared with those of sPLA(2)-X(+/+) littermates, had significant reduction in ovalbumin-induced infiltration by CD4(+) and CD8(+) T cells and eosinophils, goblet cell metaplasia, smooth muscle cell layer thickening, subepithelial fibrosis, and levels of T helper type 2 cell cytokines and eicosanoids. These data direct attention to sPLA(2)-X as a novel therapeutic target for asthma.

Reversal of allergen-induced airway remodeling by CysLT1 receptor blockade.

RATIONALE: Airway inflammation in asthma is accompanied by structural changes, including goblet cell metaplasia, smooth muscle cell layer thickening, and subepithelial fibrosis. This allergen-induced airway remodeling can be replicated in a mouse asthma model. OBJECTIVES: The study goal was to determine whether established airway remodeling in a mouse asthma model is reversible by administration of the cysteinyl leukotriene (CysLT)1 receptor antagonist montelukast, the corticosteroid dexamethasone, or the combination montelukast + dexamethasone. METHODS: BALB/c mice, sensitized by intraperitoneal ovalbumin (OVA) as allergen, received intranasal OVA periodically Days 14-73 and montelukast or dexamethasone or placebo from Days 73-163. MEASUREMENTS AND MAIN RESULTS: Allergen-induced trafficking of eosinophils into the bronchoalveolar lavage fluid and lung interstitium and airway goblet cell metaplasia, smooth muscle cell layer thickening, and subepithelial fibrosis present on Day 73 persisted at Day 163, 3 mo after the last allergen challenge. Airway hyperreactivity to methacholine observed on Day 73 in OVA-treated mice was absent on Day 163. In OVA-treated mice, airway eosinophil infiltration and goblet cell metaplasia were reduced by either montelukast or dexamethasone alone. Montelukast, but not dexamethasone, reversed the established increase in airway smooth muscle mass and subepithelial collagen deposition. By immunocytochemistry, CysLT1 receptor expression was significantly increased in airway smooth muscle cells in allergen-treated mice compared with saline-treated controls and was reduced by montelukast, but not dexamethasone, administration. CONCLUSIONS: These data indicate that established airway smooth muscle cell layer thickening and subepithelial fibrosis, key allergen-induced airway structural changes not modulated by corticosteroids, are reversible by CysLT1 receptor blockade therapy.

Augmented lung injury due to interaction between hyperoxia and mechanical ventilation.

OBJECTIVE: Mechanical overdistension and hyperoxia can independently cause lung injury, yet little is known about their combined effects. We hypothesized that hyperoxia exacerbates lung injury caused by large tidal volume ventilation. DESIGN: Experimental study. SETTING: University laboratory. SUBJECTS: Anesthetized, paralyzed rabbits. INTERVENTIONS: In experiment 1, 12 rabbits were ventilated with 25 mL/kg tidal volumes at positive end-expiratory pressure of 0 cm H2O for 4 hrs with either hyperoxia (HO; FiO2 = 0.5) or normoxia (NO; FiO2 = 0.21). In experiment 2, a separate group of animals were randomized to one of four groups to assess the interaction of tidal volume and inspired oxygen concentration on potential mediators of injury after 2 hrs of ventilation, before significant injury occurs: a) NO+normal tidal volume (NV; VT = 10 mL/kg); b) HO+NV; c) NO+high tidal volume (HV; VT = 25 mL/kg); d) HO+HV (n = 3 per group). MEASUREMENTS AND MAIN RESULTS: : In the first study, HO compared with the NO group had significantly reduced PaO2/FiO2 ratio (320 +/- 110 vs. 498 +/- 98, p = .014) and increased lung injury scores at 4 hrs. Hyperoxia also significantly increased polymorphonuclear leukocytes, growth-related oncogene-alpha (2073 +/- 535 vs. 463 +/- 236 pg/mL, p = .02), and monocyte chemotactic protein-1 (7517 +/- 1612 vs. 2983 +/- 1289 pg/mL, p = .05) concentrations in bronchoalveolar lavage fluid. The second study showed increased alveolar-capillary permeability to a 70-kD fluorescent-labeled dextran only in response to the combination of both HO and HV. Chemokines and bronchoalveolar lavage fluid neutrophils were elevated in both HV groups; however, hyperoxia did not further increase chemokine or neutrophil counts over normoxia. No difference in lipid peroxidation was seen between groups. CONCLUSIONS: Moderate hyperoxia exacerbates lung injury in a large tidal volume model of ventilator-induced lung injury. The mechanism by which this occurs is not mediated by increased production of CXC chemokines or lipid peroxidation.

Liver hemostasis with high-intensity ultrasound: repair and healing.

OBJECTIVE: Previous studies have shown that high-intensity focused ultrasound can effectively control bleeding from injuries of liver, spleen, and blood vessels. This study investigated long-term hemostasis and tissue repair after high-intensity focused ultrasound treatment in liver. METHODS: A total of 21 rabbits were randomly assigned to 2 groups: high-intensity focused ultrasound treatment (n = 14) and sham treatment (n = 7). All animals had sterile laparotomy and liver exposure. The high-intensity focused ultrasound-treated animals received liver incisions, 20 to 25 mm long and 4 to 6 mm deep, followed immediately by high-intensity focused ultrasound application until complete hemostasis was achieved. After recovery, sonographic images, blood samples, and histologic samples were collected immediately and on days 1, 3, 7, 14, 28, and 60 after treatment. RESULTS: All 14 liver injuries were hemostatic after an average +/- SD of 78 +/- 44 seconds of high-intensity focused ultrasound application, with no rebleeding at any time point after the treatment. Subsequent blood analysis showed no significant difference in serial hematologic or coagulation measures between the high-intensity focused ultrasound and sham groups. Alanine aminotransferase and aspartate aminotransferase levels increased immediately after surgery by as much as 285% up to day 3 and returned to normal values by day 7. Hematocrit and white blood cell counts showed no statistically significant difference from normal values at all time points. Histologic examination up to 60 days after treatment revealed scarring and liver tissue regeneration at the treatment site. CONCLUSIONS: High-intensity focused ultrasound appears to provide long-lasting hemostasis of acute liver injury. Healing and repair mechanisms after high-intensity focused ultrasound application appear to be intact.

Chemogenomics with peptide secondary structure mimetics.

There is increasing evidence that redox regulation of transcription, particularly activator protein-1 (AP-1) and nuclear factor kappa B (NF-kappaB), is important in inflammatory diseases. Human thioredoxin (TRX), a member of the oxidoreductase superfamily, was initially identified, as a factor which augments the production of interleukin-2 receptor alpha (IL-2R alpha) in human T-cell lymphotropic virus type 1 (HTLV-1) infected patient T-cells. Substrates for the redox activity of TRX bind the active site cleft in extended strand structure. The rapid generation of high numbers of peptide secondary structure mimetics through solid-phase synthesis is a key technology for the identification of pharmaceutical leads based on such protein-peptide interactions. In this manuscript, we describe a chemogenomic approach utilizing an extended strand templated library to develop small molecule inhibitors to validate oxidoreductase molecular targets in a murine asthma model.

A broad-spectrum caspase inhibitor attenuates allergic airway inflammation in murine asthma model.

Asthma is characterized by acute and chronic airway inflammation, and the severity of the airway hyperreactivity correlates with the degree of inflammation. Many of the features of lung inflammation observed in human asthma are reproduced in OVA-sensitized/challenged mice. T lymphocytes, particularly Th2 cells, are critically involved in the genesis of the allergic response to inhaled Ag. In addition to antiapoptotic effects, broad-spectrum caspase inhibitors inhibit T cell activation in vitro. We investigated the effect of the broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), on airway inflammation in OVA-sensitized/challenged mice. OVA-sensitized mice treated with z-VAD-fmk immediately before allergen challenge showed marked reduction in inflammatory cell infiltration in the airways and pulmonary blood vessels, mucus production, and Th2 cytokine production. We hypothesized that the caspase inhibitor prevented T cell activation, resulting in the reduction of cytokine production and eosinophil infiltration. Treatment with z-VAD-fmk in vivo prevented subsequent T cell activation ex vivo. We propose that caspase inhibitors may offer a novel therapeutic approach to T cell-dependent inflammatory airway diseases.

Chemogenomic identification of Ref-1/AP-1 as a therapeutic target for asthma.

Asthma is characterized by an oxidantantioxidant imbalance in the lungs leading to activation of redox-sensitive transcription factors, nuclear factor kappaB (NF-kappaB), and activator protein-1 (AP-1). To develop therapeutic strategies for asthma, we used a chemogenomics approach to screen for small molecule inhibitor(s) of AP-1 transcription. We developed a beta-strand mimetic template that acts as a reversible inhibitor (pseudosubstrate) of redox proteins. This template incorporates an enedione moiety to trap reactive cysteine nucleophiles in the active sites of redox proteins. Specificity for individual redox factors was achieved through variations in X and Y functionality by using a combinatorial library approach. A limited array (2 x 6) was constructed where X was either NHCH(3) or NHCH(2) Ph and Y was methyl, phenyl, m-cyanophenyl, m-nitrophenyl, m-acetylaniline, or m-methylbenzoate. These analogs were evaluated for their ability to inhibit transcription in transiently transfected human lung epithelial A549 cells from either an AP-1 or NF-kappaB reporter. A small-molecule inhibitor, PNRI-299, was identified that selectively inhibited AP-1 transcription (IC(50) of 20 microM) without affecting NF-kappaB transcription (up to 200 microM) or thioredoxin (up to 200 microM). The molecular target of PNRI-299 was determined to be the oxidoreductase, redox effector factor-1 by an affinity chromatography approach. The selective redox effector factor-1 inhibitor, PNRI-299, significantly reduced airway eosinophil infiltration, mucus hypersecretion, edema, and IL-4 levels in a mouse asthma model. These data validate AP-1 as an important therapeutic target in allergic airway inflammation.

A small molecule inhibitor of redox-regulated NF-kappa B and activator protein-1 transcription blocks allergic airway inflammation in a mouse asthma model.

An oxidant/antioxidant imbalance is seen in the lungs of patients with asthma. This oxidative stress in asthmatic airways may lead to activation of redox-sensitive transcription factors, NF-kappaB and AP-1. We examined the effect of the small molecule inhibitor of redox-regulated NF-kappaB and AP-1 transcription, MOL 294 on airway inflammation and airway hyperreactivity (AHR) in a mouse model of asthma. MOL 294 is a potent nonpeptide inhibitor of NF-kappaB and AP-1 based upon a beta-strand template that binds to and inhibits the cellular redox protein thioredoxin. BALB/c mice after i.p. OVA sensitization (day 0) were challenged with intranasal OVA on days 14, 25, 26, and 27. MOL 294, administered intranasal on days 25-27, blocked the airway inflammatory response to OVA assessed 24 h after the last OVA challenge on day 28. MOL 294 reduced eosinophil, IL-13, and eotaxin levels in bronchoalveolar lavage fluid and airway tissue eosinophilia and mucus hypersecretion. MOL 294 also decreased AHR in vivo to methacholine. These results support redox-regulated transcription as a therapeutic target in asthma and demonstrate that selective inhibitors can reduce allergic airway inflammation and AHR.

Treatment of uterine leiomyosarcoma in a xenograft nude mouse model using high-intensity focused ultrasound: a potential treatment modality for recurrent pelvic disease.

OBJECTIVE: The objective was to test the efficacy of high-intensity focused ultrasound (HIFU) for treatment of uterine leiomyosarcoma in a Xenograft nude mouse model. METHODS: A total of 65 athymic nude mice were inoculated subcutaneously with 5 to 7 x 10(6) ELT-5B cells, a uterine leiomyosarcoma cell line derived from the Eker rat. Thirty animals showed tumor growth. The tumor volume was measured transcutaneously once a week. Animals were randomly assigned to three groups: HIFU treatment (n = 17), sham treatment (n = 7), and control (n = 6). A HIFU device, operating at a frequency of 2.0 MHz and an intensity of 2000 W/cm(2), was used for treatment. RESULTS: Within 3 weeks of a single HIFU treatment, 100% reduction in tumor volume was observed in all animals, except one. A second HIFU treatment was applied to that animal, resulting in 100% reduction in tumor volume. The tumors in the sham-treated animals continued to grow at a similar rate to that of the control group to approximately 500% of the tumor volume at the time of treatment. All animals were monitored for a maximum of 3 months. No metastasis was observed in the HIFU-treated animals. Histological examination confirmed a complete tumor disappearance after HIFU treatment. CONCLUSION: We have shown that HIFU can effectively treat uterine leiomyosarcoma tumors inoculated in Xenograft nude mice, demonstrating HIFU's potential use for treatment of recurrent uterine leiomyosarcoma.

A role for cysteinyl leukotrienes in airway remodeling in a mouse asthma model.

Airway inflammation and remodeling in chronic asthma are characterized by airway eosinophilia, hyperplasia of goblet cells and smooth muscle, and subepithelial fibrosis. We examined the role of leukotrienes in a mouse model of allergen-induced chronic lung inflammation and fibrosis. BALB/c mice, after intraperitoneal ovalbumin (OVA) sensitization on Days 0 and 14, received intranasal OVA periodically Days 14-75. The OVA-treated mice developed an extensive eosinophil and mononuclear cell inflammatory response, goblet cell hyperplasia, and mucus occlusion of the airways. A striking feature of this inflammatory response was the widespread deposition of collagen beneath the airway epithelial cell layer and also in the lung interstitium in the sites of leukocytic infiltration that was not observed in the saline-treated controls. The cysteinyl leukotriene(1) (CysLT(1)) receptor antagonist montelukast significantly reduced the airway eosinophil infiltration, mucus plugging, smooth muscle hyperplasia, and subepithelial fibrosis in the OVA-sensitized/challenged mice. The presence of Charcot-Leyden-like crystals in airway macrophages and the increased interleukin (IL)-4 and IL-13 mRNA expression in lung tissue and protein in BAL fluid seen in OVA-treated mice were also inhibited by CysLT(1) receptor blockade. These data suggest an important role for cysteinyl leukotrienes in the pathogenesis of chronic allergic airway inflammation with fibrosis.