Targeting the blood-brain barrier disruption in hypertension by ALK5/TGF-Β type I receptor inhibitor SB-431542 and dynamin inhibitor dynasore.

INTRODUCTION: In this study, we aimed to target two molecules, transforming growth factor-beta (TGF-ß) and dynamin to explore their roles in blood-brain barrier (BBB) disruption in hypertension. METHODS: For this purpose, angiotensin (ANG) II-induced hypertensive mice were treated with SB-431542, an inhibitor of the ALK5/TGF-ß type I receptor, and dynasore, an inhibitor of dynamin. Albumin-Alexa fluor 594 was used to assess BBB permeability. The alterations in the expression of claudin-5, caveolin (Cav)-1, glucose transporter (Glut)-1, and SMAD4 in the cerebral cortex and the hippocampus were evaluated by quantification of immunofluorescence staining intensity. RESULTS: ANG II infusion increased BBB permeability to albumin-Alexa fluor 594 which was reduced by SB-431542 (P < 0.01), but not by dynasore. In hypertensive animals treated with dynasore, claudin-5 immunofluorescence intensity increased in the cerebral cortex and hippocampus while it decreased in the cerebral cortex of SB-431542 treated hypertensive mice (P < 0.01). Both dynasore and SB-431542 prevented the increased Cav-1 immunofluorescence intensity in the cerebral cortex and hippocampus of hypertensive animals (P < 0.01). SB-431542 and dynasore decreased Glut-1 immunofluorescence intensity in the cerebral cortex and hippocampus of mice receiving ANG II (P < 0.01). SB-431542 increased SMAD4 immunofluorescence intensity in the cerebral cortex of hypertensive animals, while in the hippocampus a significant decrease was noted by both SB-431542 and dynasore (P < 0.01). CONCLUSION: Our data suggest that inhibition of the TGFß type I receptor prevents BBB disruption under hypertensive conditions. These results emphasize the therapeutic potential of targeting TGFß signaling as a novel treatment modality to protect the brain of hypertensive patients.

Development of a Rapid and Sensitive IDH1/2 Mutation Detection Method for Glial Tumors and a Comparative Mutation Analysis of 236 Glial Tumor Samples.

BACKGROUND: The presence of mutations in the isocitrate dehydrogenase 1 and 2 genes (IDH1/2) in glioma tumors is correlated with good prognosis upon standard-of-care treatment. Therefore, information on whether the glioma tumor has IDH1/2 mutations could be used in the correct diagnosis and management of glial tumors. The two most common techniques used to detect IDH1/2 mutations, immunohistochemistry (IHC) and Sanger sequencing, are prone to missing these mutations, especially if the tumor cells that carry the mutations constitute a small minority of the tumor itself. OBJECTIVES: We developed and validated a rapid method (3-mismatch-amplification refractory mutation system [3m-ARMS]) that can be used for pre-, intra- and postoperative detection of the most common IDH1/2 mutations in glial tumors with high specificity and sensitivity. We also conducted a comprehensive IDH1/2 mutation analysis in 236 glial tumor samples comparing 3m-ARMS, IHC and Sanger sequencing. METHODS: 3m-ARMS was optimized and validated for the specific and sensitive detection of the most common IDH1 and IDH2 mutations. We then analyzed 236 glial tumor samples for the presence of IDH1/2 mutations using 3m-ARMS, Sanger sequencing and IHC techniques. We then analyzed and compared the results, evaluating the diagnostic and screening potential of 3m-ARMS. RESULTS: Comparison of the three techniques used in the mutation analysis showed that 3m-ARMS-based IDH1/2 mutation detection was superior to IHC and Sanger sequencing-based IDH1/2 mutation detection in terms of accuracy, specificity and sensitivity, especially for tumor samples in which only a small minority of the cell population carried the mutation. 3m-ARMS could detect the presence of femtogram levels of IDH1/2 mutant DNA in DNA samples in which the mutant DNA-to-wild-type DNA ratio was as low as 1:100,000. CONCLUSION: Sanger sequencing and IHC-based methods have shortcomings when detecting mutations in glial tumors so can miss IDH1/2 mutations in glial tumors when used alone without proper modifications. 3m-ARMS-based mutation detection is fast and simple with potential for use as a diagnostic test for the majority of hot spot mutations in IDH1/2 genes. It can detect IDH1/2 mutations within an hour so can be adapted for intraoperative diagnosis.