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1.
STAR Protoc ; 5(3): 103170, 2024 Jul 04.
Article in English | MEDLINE | ID: mdl-38968077

ABSTRACT

Three-dimensional (3D) imaging of vascular networks is essential for the investigation of vascular patterning and organization. Here, we present a step-by-step protocol for the 3D visualization of the vasculature within whole-mount preparations of the mouse intestinal muscularis propria layer. We then detail the quantitative analysis of the resulting images for parameters such as vessel density, vessel diameter, the number of endothelial cells, and proliferation. The protocol can be easily extended to study cell-cell interactions such as neuro-vascular or immune-vascular interactions. For complete details on the use and execution of this protocol, please refer to Schrenk et al.1.

2.
Nat Commun ; 14(1): 1929, 2023 04 06.
Article in English | MEDLINE | ID: mdl-37024491

ABSTRACT

Activating non-inherited mutations in the guanine nucleotide-binding protein G(q) subunit alpha (GNAQ) gene family have been identified in childhood vascular tumors. Patients experience extensive disfigurement, chronic pain and severe complications including a potentially lethal coagulopathy termed Kasabach-Merritt phenomenon. Animal models for this class of vascular tumors do not exist. This has severely hindered the discovery of the molecular consequences of GNAQ mutations in the vasculature and, in turn, the preclinical development of effective targeted therapies. Here we report a mouse model expressing hyperactive mutant GNAQ in endothelial cells. Mutant mice develop vascular and coagulopathy phenotypes similar to those seen in patients. Mechanistically, by transcriptomic analysis we demonstrate increased mitogen activated protein kinase signaling in the mutant endothelial cells. Targeting of this pathway with Trametinib suppresses the tumor growth by reducing vascular cell proliferation and permeability. Trametinib also prevents the development of coagulopathy and improves mouse survival.


Subject(s)
Melanoma , Uveal Neoplasms , Vascular Neoplasms , Animals , Mice , GTP-Binding Protein alpha Subunits/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Endothelial Cells/metabolism , Apoptosis , Melanoma/genetics , Uveal Neoplasms/genetics , Mutation , Disease Models, Animal , Mitogen-Activated Protein Kinase Kinases/metabolism , Cell Line, Tumor
3.
Clin Exp Allergy ; 49(3): 317-330, 2019 03.
Article in English | MEDLINE | ID: mdl-30353972

ABSTRACT

BACKGROUND: Recent studies have demonstrated that Th2 responses have the ability to antagonize Th17 responses. In mouse models of allergic asthma, blockade of Th2-effector cytokines results in elaboration of Th17 responses and associated increases in pulmonary neutrophilia. While these can be controlled by simultaneous blockade of Th17-associated effector cytokines, clinical trials of anti-IL-17/IL-17RA blocking therapies have demonstrated increased of risk of bacterial and fungal infections. Identification of minimally effective doses of cytokine-blocking therapies with the goal of reducing the potential emergence of infection-related complications is a translationally relevant goal. OBJECTIVE: In the current report, we examine whether combined blockade of IL-13 and IL-17A, at individually sub-therapeutic levels, can limit the development of allergic asthma while sparing expression of IL-17A-associated anti-microbial effectors. METHODS: House dust mite was given intratracheally to A/J mice. Anti-IL-13 and anti-IL-17A antibodies were administered individually, or concomitantly at sub-therapeutic doses. Airway hyper-reactivity, lung inflammation, magnitude of Th2- and Th17-associated cytokine production and expression of IL-13- and IL-17A-induced genes in the lungs was assessed. RESULTS: Initial dosing studies identified sub-therapeutic levels of IL-13 and IL-17A blocking mAbs that have a limited effect on asthma parameters and do not impair responses to microbial products or infection. Subsequent studies demonstrated that combined sub-therapeutic dosing with IL-13 and IL-17A blocking mAbs resulted in significant improvement in airway hyperresponsiveness (AHR) and expression of IL-13-induced gene expression. Importantly, these doses neither exacerbated nor inhibited production of Th17-associated cytokines, or IL-17A-associated gene expression. CONCLUSION: This study suggests that combining blockade of individual Th2 and Th17 effector cytokines, even at individually sub-therapeutic levels, may be sufficient to limit disease development while preserving important anti-microbial pathways. Such a strategy may therefore have reduced potential for adverse events associated with blockade of these pathways.


Subject(s)
Antibodies, Blocking/pharmacology , Asthma/immunology , Interleukin-13/antagonists & inhibitors , Interleukin-17/antagonists & inhibitors , Th17 Cells/immunology , Th2 Cells/immunology , Animals , Asthma/pathology , Cytokines/immunology , Disease Models, Animal , Interleukin-13/immunology , Interleukin-17/immunology , Mice , Pyroglyphidae/immunology , Th17 Cells/pathology , Th2 Cells/pathology
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