PMC:4608092 / 43456-47358 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4608092","sourcedb":"PMC","sourceid":"4608092","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4608092","text":"Effects of Expression of the RNF213 R4810K Ortholog Rnf213 R4757K on Cerebral Angiogenesis In Vivo\nOur in vitro data strongly suggest that RNF213 R4810K lowers angiogenic activity in ECs when it is induced by environmental stimuli, such as IFNs. Therefore, we investigated the effects of ablation or upregulation of RNF213 R4810K in vivo using various genetically modified mice. These mouse strains involved ablation of Rnf213 (KO)17 and Tg mice, which overexpresses Rnf213 (R4757K or WT) in ECs (EC: EC-Mut-Tg and EC-WT-Tg) or SMCs (SMC: SMC-Mu-Tg) (Figure2). Tissue-specific upregulation of RNF213 was confirmed in Tg mice in ECs and SMCs (Figure7). To induce cerebral angiogenesis, mice at 3 weeks of age were exposed to hypoxia (8% O2 for 2 weeks). At the end of exposure, MRA was conducted in 3 mice for each strain. Exposure to hypoxia failed to induce angiogenesis in EC-Mut-Tg mice, whereas upregulation of Rnf213 WT in ECs, upregulation of Rnf213 R4757K in SMCs, and null Rnf213 or WT significantly induced angiogenesis (Figure8). However, we could not find any stenotic lesions, moyamoya vessels, or lesions indicative of cerebral infarction in any mice with different genotypes (Figure9).\nFigure 17 Tissue-specific upregulation of RNF213 in EC-Tg and SMC-Tg mice. Protein extraction from purified ECs from lungs of EC-Mut Tg and WT mice (left panel) and protein extraction from aorta of SMC-Mut Tg and WT mice (right panel) were immunoblotted. β-tubulin served as the loading control. Similar results were obtained from 3 independent experiments. ECs indicates endothelial cells; SMC, smooth muscle cells; Tg, transgenic; WT, wild type.\nFigure 18 Suppressive effect of Rnf213 mutant upregulation in ECs on angiogenesis in vivo. A, Representative images of GLUT-1-stained sections of cerebral cortex of EC-Mut Tg, EC-WT Tg, SMC-Mut Tg, KO, and WT mice under conditions of normoxia (N) and hypoxia (H). B, Quantified result of cerebral microvessels (left panel). A column with a bar represents mean±SD of the number of cerebral microvessels/mm2 from 6 mice per group. In the hypoxia condition, there was a significant difference in the number of cerebral microvessels among 5 genotypes using the nonparametric method, Kruskal–Wallis 1-way ANOVA (P=0.036), but not in the normoxia condition (P=0.41). *P\u003c0.05 according to Mann–Whitney U test compared with normoxia condition. Two-way ANOVA method was conducted for microvessel formation between genotypes and treatment with interaction term. Results and parameter estimates are described in table (right panel). Regression models are described as (microvessels)=(intercept)+α×[Genotype: EC-Mut-Tg]+β×[Genotype: EC-WT-Tg]+γ×[Genotype: SMC-Mut-Tg]+δ×[Genotype: KO]+ε×[Treatment]+ζ×[interaction: EC-Mut-Tg]+η×[interaction: EC-WT-Tg]+θ×[interaction: SMC-Mut-Tg]+ι×[interaction: KO]. Treatment (hypoxia) significantly induced the number of microvessels/mm2 (P\u003c0.001), whereas genotype did not (P=0.09). Interaction between genotype and treatment was significant (P=0.02). The coefficient on EC-Mut-Tg (ζ) was negative, suggesting that this genotype did not increase microvessels (P=0.002). #P\u003c0.05 according to 2-way ANOVA. ECs indicates endothelial cells; GLUT-1, glucose transporter; KO, knockout; SMC, smooth muscle cells; Tg, transgenic; WT, wild type.\nFigure 19 Representative MRI image of brain of EC-Mut Tg, EC-WT Tg, SMC-Mut Tg, KO, and WT mice with hypoxia. MRA (upper panel) represents MRA images. No stenotic lesions and moyamoya vessels were detected in brain. T2 (lower panel) represents T2-weighted images. No infarction was detected in brains. Absence of stenotic lesion, moyamoya vessel, and infarction was also confirmed in other 2 mice in each genotype. EC indicates endothelial cell; KO, knockout; MRA, magnetic resonance angiography; MRI, magnetic resonance imaging; SMC, smooth muscle cells; Tg, transgenic; WT, wild type.\n\nD","divisions":[{"label":"Title","span":{"begin":0,"end":98}},{"label":"Figure caption","span":{"begin":1200,"end":1650}},{"label":"Figure caption","span":{"begin":1649,"end":3313}},{"label":"Figure caption","span":{"begin":3312,"end":3901}}],"tracks":[{"project":"2_test","denotations":[{"id":"26126547-23410753-55294645","span":{"begin":431,"end":433},"obj":"23410753"}],"attributes":[{"subj":"26126547-23410753-55294645","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec93b7","default":true}]}]}}