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K. M. Janisch, J. M. Kasanuki, J. Tosi, Y. S. Grossman, R. J. Davis, S. H. Tsang; Biochemical Analysis of the Hypoxia Response in HIF1alpha Overexpressing Mice. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3412.
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© ARVO (1962-2015); The Authors (2016-present)
The VHL protein plays a role in the hypoxia pathway by regulating the degradation of HIF1 as part of the E3 ubiquitin ligase. Under hypoxic conditions, HIF1 is not hydroxylized for degradation and upregulates gene products relevant for angiogenesis (VEGF), erythropoiesis (EPO), regulation of extracellular pH (CA IX), cell proliferation/survival (GSK3β, cyclin D1, COX2) and nitric oxide signaling (eNOS). These events have been extensively studied for clear-cell renal cell carcinoma occurring in the rare von Hippel-Lindau (VHL) disease, which derives from mutations in the VHL tumor suppressor protein. These VHL mutations increase HIF1 levels due to the failure of degradation. Increased HIF1 levels transcribe above mentioned gene products. The influence of VHL on HIF1 regulation is part of the hypoxia switch. Presently, there are no data published on mice with VHL eye phenotypes. A conditional knockout of VHL that produces an eye phenotype in mice can contribute to the understanding of VHL in retinal hypoxia.
A conditional VHLflox/flox mouse was generated producing a retinal phenotype (see 2009 ARVO abstract "Abnormal retinal vascular development in von Hippel-Lindau (Vhl) mutant mice" by J.M. Kasanuki et al.). Live fundus imaging was performed through the lens of the eye under a surgical microscope. For Western blot analysis P3, P6, P14 and P28 old mutant and age-matched litter-mates as well as C57/BL6 control mice were chosen. Retinae were extracted for cytosolic and nuclear protein fractions. The fractions were subjected to SDS-PAGE electrophoresis (standardized gel loading according to total protein content). Proteins were transferred on nitrocellulose and probed with the following antibodies: anti-HIF1, anti-eNOS, anti-GSK3β, anti-EPO, anti-COX2, anti-cyclin D1 and anti-CA IX.
Live fundus images of VHLflox/flox mice showed disorganized vascularization of the retinae. Western blot analysis confirmed the increase in the transcription factor HIF1 in both the cytosolic as well as the nuclear fraction of the mutant mice. Results for Western blot analysis with anti-eNOS, anti-GSK3β, anti-EPO, anti-COX2, anti-cyclin D1 and anti-CA IX are in progress.
The preliminary results show the upregulation of the transcription factor HIF1. For upcoming data, we expect the upregulation for the downstream targets in these mice. With all the biochemical data and evaluation of this conditional VHLflox/flox mouse, there might be a valid model to investigate retinal hypoxia and vascular development.
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