IFN-γ drives inflammatory bowel disease pathogenesis through VE-cadherin-directed vascular barrier disruption.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder with rising incidence. Diseased tissues are heavily vascularized. Surprisingly, the pathogenic impact of the vasculature in IBD and the underlying regulatory mechanisms remain largely unknown. IFN-γ is a major cytokine in IBD pathogenesis, but in the context of the disease, it is almost exclusively its immune-modulatory and epithelial cell-directed functions that have been considered. Recent studies by our group demonstrated that IFN-γ also exerts potent effects on blood vessels. Based on these considerations, we analyzed the vessel-directed pathogenic functions of IFN-γ and found that it drives IBD pathogenesis through vascular barrier disruption. Specifically, we show that inhibition of the IFN-γ response in vessels by endothelial-specific knockout of IFN-γ receptor 2 ameliorates experimentally induced colitis in mice. IFN-γ acts pathogenic by causing a breakdown of the vascular barrier through disruption of the adherens junction protein VE-cadherin. Notably, intestinal vascular barrier dysfunction was also confirmed in human IBD patients, supporting the clinical relevance of our findings. Treatment with imatinib restored VE-cadherin/adherens junctions, inhibited vascular permeability, and significantly reduced colonic inflammation in experimental colitis. Our findings inaugurate the pathogenic impact of IFN-γ-mediated intestinal vessel activation in IBD and open new avenues for vascular-directed treatment of this disease.

Noninvasive Bioluminescence Imaging of AKT Kinase Activity in Subcutaneous and Orthotopic NSCLC Xenografts: Correlation of AKT Activity with Tumor Growth Kinetics.

Aberrant signaling through the AKT kinase mediates oncogenic phenotypes including cell proliferation, survival, and therapeutic resistance. Here, we utilize a bioluminescence reporter for AKT kinase activity (BAR) to noninvasively assess the therapeutic efficacy of the EGFR inhibitor erlotinib in KRAS-mutated lung cancer therapy. A549 non-small cell lung cancer cell line, engineered to express BAR, enabled the evaluation of compounds targeting the EGFR/PI3K/AKT pathway in vitro as well as in mouse models. We found that erlotinib treatment of resistant A549 subcutaneous and orthotopic xenografts resulted in significant AKT inhibition as determined by an 8- to 13-fold (P < .0001) increase in reporter activity 3 hours after erlotinib (100 mg/kg) administration compared to the control. This was confirmed by a 25% (P < .0001) decrease in pAKT ex vivo and a decrease in tumor growth. Treatment of the orthotopic xenograft with varying doses of erlotinib (25, 50, and 100 mg/kg) revealed a dose- and time-dependent increase in reporter activity (10-, 12-, and 23-fold). Correspondingly, a decrease in phospho-AKT levels (0%, 16%, and 28%, respectively) and a decrease in the AKT dependent proliferation marker PCNA (0%, 50%, and 50%) were observed. We applied µ-CT imaging for noninvasive longitudinal quantification of lung tumor load which revealed a corresponding decrease in tumor growth in a dose-dependent manner. These findings demonstrate the utility of BAR to noninvasively monitor AKT activity in preclinical studies in response to AKT modulating agents. These results also demonstrate that BAR can be applied to study drug dosing, drug combinations, and treatment efficacy in orthotopic mouse lung tumor models.