semaxinib and Inflammation

We used inflammatory breast cancer (IBC) as a model disease to investigate biological changes associated with an antiangiogenesis agent, SU5416, combined with doxorubicin.. Patients with stage IIIB or IV IBC were treated neoadjuvantly with the combination of SU5416 and doxorubicin for induction therapy. The dose of SU5416 (administered on days 1 and 4, every 3 weeks) and doxorubicin (administered on day 1 every 3 weeks) were escalated in cohorts of three patients starting at 110 and 60 mg/m2, respectively, for a total of five cycles leading up to mastectomy. Patients underwent serial assessment (pharmacokinetic sampling, biopsy of breast, tumor blood flow dynamic contrast-enhanced magnetic resonance imaging, plasma angiogenesis, and endothelial cell damage markers) prior to treatment, at the end of cycles no. 2 and no. 5, and after mastectomy.. Eighteen patients were enrolled; neutropenia was dose-limiting, and overall median survival was not reached (50 months of study follow-up). Four patients (22%) experienced congestive heart failure, which resolved and were likely attributable to a smaller volume of distribution and higher Cmax of doxorubicin in combination with SU5416. We did observe a significant decline in tumor blood flow using Kep calculated by Brix (pretreatment versus post-cycle no. 5; P = 0.033), trend for a decline in tumor microvessel density after treatment, and low baseline levels of soluble intracellular adhesion molecule were associated with improved event-free survival.. This study showed evidence of an unfavorable cardiac interaction between SU5416 and doxorubicin, which prohibits further investigation of this combination. However, this study supports the importance of using IBC as a model for investigating angiogenesis inhibitors.

Topics: Adult; Aged; Angiogenesis Inhibitors; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Disease-Free Survival; Doxorubicin; Female; Humans; Indoles; Inflammation; Male; Maximum Tolerated Dose; Middle Aged; Neovascularization, Pathologic; Pyrroles; Treatment Outcome

Objective- Macrophages participate in the pathogenesis of pulmonary arterial hypertension (PAH). Lgmn (Legumain), a newly discovered cysteine proteinase belonging to the C13 peptidase family, is primarily expressed in macrophages; however, its roles in PAH remain unknown. Approach and Results- Herein, Lgmn was upregulated in lung tissues of PAH mice subjected to hypoxia plus SU5416 and PAH rats challenged with monocrotaline. Global Lgmn ablation and macrophage-specific ablation alleviated PAH compared with wild-type mice, evident from a reduction in right ventricular systolic pressure, the ratio of the right ventricular wall to the left ventricular wall plus the septum, the pulmonary vascular media thickness, and pulmonary vascular muscularization. Increased expression of ECM (extracellular matrix) proteins was correlated with MMP (matrix metalloproteinase)-2 activation and TGF (transforming growth factor)-β1 signaling in the PAs. Although Lgmn did not affect inflammatory cell infiltration and PA smooth muscle cell proliferation, it drove increased the synthesis of ECM proteins via MMP-2 activation. MMP-2 hydrolyzed the TGF-β1 precursor to the active form. An Lgmn-specific inhibitor markedly ameliorated PAH. Clinically, serum Lgmn levels were closely associated with the severity of idiopathic PAH. Conclusions- Our results indicate that Lgmn inhibition could be an effective strategy for preventing or delaying PAH.

Topics: Animals; Caspase Inhibitors; Cysteine Endopeptidases; Extracellular Matrix Proteins; Female; Follow-Up Studies; Humans; Hypertension, Pulmonary; Hypoxia; Indoles; Inflammation; Lung; Macrophages; Male; Matrix Metalloproteinase 2; Mice; Middle Aged; Monocrotaline; Pyrroles; Rats; Severity of Illness Index; Signal Transduction; Transforming Growth Factor beta1; Vascular Remodeling

Severe pulmonary arterial hypertension (PAH) is an incurable disease whose exact mechanisms remain unknown. However, growing evidence highlights the role of inflammation and endothelin (ET) signaling. The lack of reliable models makes it difficult to investigate the pathophysiology of this disease. Our aim was therefore to develop a mouse model of severe PAH closely mimicking the human condition to explore the role of interleukin-6 (IL-6), and ET signaling in advanced PAH progression.. Young male SV129 mice received vascular endothelial growth factor receptor inhibitor (SU5416) three times a week and were exposed to hypoxia (10% O2) for three weeks. Molecular analysis and histological assessment were examined using real-time PCR, Western blot and immunostaining, respectively.. The developed murine model presented important characteristics of severe PAH in human: concentric neointimal wall thickening, plexogenic lesions, recruitment of macrophages, and distal arteriolar wall muscularization. We detected an increase of IL-6 production and a stronger macrophage recruitment in adventitia of remodeled arterioles developing plexogenic lesions. Moreover, ET-1 and ET receptor A were up-regulated in lung lysates and media of remodeled arterioles. Recombinant IL-6 stimulated the proliferation and regulated endothelial cells in increasing ET-1 and decreasing ET receptor B.. These data describe a murine model, which displays the most important features of human severe PAH. We assume that inflammation, particularly IL-6 regulating ET signaling, plays a crucial role in forming plexogenic lesions. This model is thus reliable and might be used for a better understanding of severe PAH progression and treatment.

Topics: Animals; Biomarkers; Body Weight; Cell Hypoxia; Cell Line; Disease Models, Animal; Endothelial Cells; Endothelin-1; Heart Rate; Humans; Hypertension, Pulmonary; Indoles; Inflammation; Interleukin-6; Lung; Male; Mice; Pulmonary Artery; Pyrroles; Receptors, Vascular Endothelial Growth Factor; Signal Transduction; Up-Regulation

NF-κB and IL-6, a NF-κB downstream mediator, play a central role in the inflammatory response of tissues. We aimed to determine the role of the classical NF-κB pathway in severe pulmonary arterial hypertension (PAH) induced by SU5416 and chronic hypoxia (SuHx) in rats. Tissue samples from patients with idiopathic PAH (iPAH) and control subjects were investigated. SuHx rats were treated from Days 1 to 3, 1 to 21, and 29 to 42 with the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) and/or from Days 1 to 21 with anti-IL-6 antibody. Nuclear staining for NF-κB, an indicator of the activation of the classical NF-κB pathway, was detected in pulmonary arterial lesions of patients with iPAH and SuHx rats. NF-κB inhibition with PDTC prevented and reduced pulmonary arterial obliteration without reducing muscularization. However, the elevated lung levels of IL-6 were not reduced in PDTC-treated SuHx animals. PDTC treatment prevented or reduced apoptosis of pulmonary artery wall cells and pulmonary arterial obliteration. IL-6 inhibition had only a partial effect on apoptosis and obliteration. Pulmonary arterial media wall thickness was not affected by any of these treatments. Preventive and therapeutic PDTC treatment promoted immune regulation by increasing the number of perivascular CD4(+) T cells, in particular regulatory T cells (early treatment), and by reducing the number of perivascular CD8(+) T lymphocytes and CD45RA(+) B lymphocytes. Therapeutic PDTC treatment further preserved right ventricular function in SuHx animals. Inhibition of NF-κB may represent a therapeutic option for pulmonary arterial obliteration via reduced vessel wall cell apoptosis and improved regulation of the immune system.

Topics: Animals; Apoptosis; CD4-Positive T-Lymphocytes; Familial Primary Pulmonary Hypertension; Humans; Hypertension, Pulmonary; Hypoxia; Indoles; Inflammation; Interleukin-6; Leukocyte Common Antigens; Lung; NF-kappa B; Pulmonary Artery; Pyrroles; Pyrrolidines; Rats; Signal Transduction; Thiocarbamates; Time Factors

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature

Chronic inflammatory airway diseases including asthma are characterized by immune dysfunction to inhaled allergens. Our previous studies demonstrated that T cell priming to inhaled allergens requires LPS, which is ubiquitously present in household dust allergens. In this study, we evaluated the role of vascular endothelial growth factor (VEGF) in the development of T cell priming and its polarization to Th1 or Th17 cells when exposed to LPS-contaminated allergens. An asthma mouse model was induced by airway sensitization with LPS-contaminated allergens and then challenged with allergens alone. Therapeutic intervention was performed during allergen sensitization. The present study showed that lung inflammation induced by sensitization with LPS-contaminated allergens was decreased in mice with homozygous disruption of the IL-17 gene; in addition, allergen-specific Th17 immune response was abolished in IL-6 knockout mice. Meanwhile, in vivo production of VEGF was up-regulated by airway exposure of LPS. In addition, airway sensitization of allergen plus recombinant VEGF induced both type 1 and type 17 Th cell (Th1 and Th17) responses. Th1 and Th17 responses induced by airway sensitization with LPS-contaminated allergens were blocked by treatment with a pan-VEGF receptor (VEGFR; VEGFR-1 plus VEGFR-2) inhibitor during sensitization. These effects were accompanied by inhibition of the production of Th1 and Th17 polarizing cytokines, IL-12p70 and IL-6, respectively. These findings indicate that VEGF produced by LPS plays a key role in activation of naive T cells and subsequent polarization to Th1 and Th17 cells.

Topics: Allergens; Animals; Apoptosis; Asthma; Immunity, Active; Immunity, Innate; Indoles; Inflammation; Interleukin-12; Interleukin-17; Interleukin-6; Lipopolysaccharides; Lung; Mice; Mice, Inbred BALB C; Mice, Knockout; Ovalbumin; Protein Kinase Inhibitors; Pyrroles; T-Lymphocytes, Helper-Inducer; Th1 Cells; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2