Progress in development of murine models with VHL-associated CNS and retinal hemangioblastomas Von Hippel-Lindau (VHL) disease is an autosomal dominant tumor predisposition syndrome that is characterized by the development of highly vascularized tumors and cysts. Loss of heterozygosity (LOH) of the VHL gene, which results in aberrant upregulation of hypoxia-inducible factors (HIFs), has been associated with tumor formation. Hemangioblastomas of the central nervous system and retina represent the most prevalent VHL-associated tumors. Until now, no VHL animal model has produced hemangioblastomas: the hallmark lesion of ocular VHL. In our study, we generated a murine model of VHL-associated hemangioblastomas by conditionally inactivating VHL in a hemangioblast population with the driver Scl-Cre-ERT2 transgenic line. Sixty-four percent (18/28) of the transgenic mice carrying the conditional allele and the Scl-Cre-ERT2 allele exhibited various retinal vascular anomalies following tamoxifen induction. Affected VHL mutant mice demonstrated retinal vascular lesions that were associated with prominent vasculature, anomalous capillary networks, hemorrhage, exudates, and localized fibrosis. Histological analyses showed hemangioblastoma-like lesions characterized by tortuous, dilated vasculature surrounded by tumorlet cells and isolated foamy stromal cells which are typically associated with hemangioblastomas. Fluorescein angiography suggested increased vascular permeability of the irregular retinal vasculature and hemangioblastoma-like lesions. VHL deletion was detected in tumorlet cells by microdissection. This is the first phenotypic recapitulation of VHL-associated RCHs in a transgenic mouse model. This model may be useful for further study of tumor hypoxia pathogenesis and for testing VHL disease and its counter-part tumor treatments. Progress in development of murine models carrying HIF2A gain-of-function mutations that exhibit similar phenotype with Pacak-Zhuang syndrome To analyze the role of HIF2A gain-of-function (GOF) mutations in the development of Pacak-Zhuang syndrome (the syndrome of paraganglioma/pheochromocytoma and polycythemia), we established a transgenic mouse model carrying the same point mutation (HIF2AA530V) in the ODD domain of HIF2A as found in patient cases. Our HIF2AA530V mutant mice showed congenital polycythemia, similar with the Pacak-Zhuang syndrome patients. We further proved that polycythemia was due to the elevated concentration of erythropoietin (EPO) in the blood. We finally confirmed that the EPO expressing cells mainly exist in the kidney in close association with peritubular capillary and proximal convoluted tubule. We also continuously measured the catecholamine level in the urine to check for the development of paraganglioma or pheochromocytoma. Our results showed that the urine catecholamine level was not elevated until 14-month old. Furthermore, the ratios of norepinephrine/epinephrine and normetanephrine/metanephrine were increased in the HIF2AA530V mutant mice compared with age-matched wild-type control. This result mimicked the immature phenotype of noradrenergic paragangliomas with high HIF2A expression. The newly established HIF2AA530V mutant mice have thus far showed promising similarity to patients with HIF2A GOF mutations in the development of Pacak-Zhuang syndrome. This animal model can be used not only for mechanistic studies of hypoxia response pathways, but also for pre-clinical drug test targeting the hypoxia response pathways. Progress in development of a cell model with combined inducible IDH1R132H and P53 mutations IDH1R132H is very commonly associated with P53 mutations in low-grade gliomas. To monitor their roles in glioma initiation and progression, we successfully generated induced pluripotent stem cells (iPS) from the peripheral mononuclear cells (PBMC) of Li-Fraumeni patients that carry germline P53 mutations (P53.C141Y) and introduced a tetracycline-regulated (tet-on) expression system of IDH1R132H. We proved that doxycycline, a tetracycline class antibiotic, efficiently induced IDH1R132H expression, and subsequently produced 2-hydroxyglutarate (2-HG), a hallmark of mutant IDH1's effect on the TCA cycle and potential indicator for gliomagenesis. The genetically modified iPS cells were further differentiated into neural progenitor cells (NPC) and subjected to doxycycline. They are currently going through passage cycles and being tested for cell integrity before differentiation continues into astrocytes. At this point in the study, we can monitor for glioma tumor-like cells and start cell metabolism studies. In addition, we plan to do in vivo work via intracranial injections using non-induced NPCs, followed by doxycycline administration into the treated mice to further our exploration of gliomagenesis.
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