Abstract
Purpose :
Von Hippel-Lindau (VHL) disease is a genetic cancer predisposition disorder, with multisystem involvement, resulting from the deletion or mutation of the VHL gene. Retinal hemangioblastomas (HBs) are large highly vascular benign tumors, and are often the first symptom leading to diagnosis. Targeted deletion of VHL in various cell types has led to models with a number of VHL-related phenotypes but has not produced a robust murine model of the large HBs observed in patients. This study seeks to establish a rigorous mouse model of retinal HBs using viral transduction of Cre recombinase in VHL floxed mice.
Methods :
“Agnostic” excision of the VHL gene in retinal tissue was achieved using virally-mediated expression of Cre recombinase and a reporter or reporter only (control) under the control of a general promoter. AAV serotypes 2, 6 or 9 were injected into the vitreous (1µl, 1010 vg). In vivo detection of vascular abnormalities was performed using Fluorescence Angiography (FA) (Micron III). Immunofluorescence (IF) in retinal wholemounts and sections was used to evaluate HB formation using the isolection IB4 for blood vessels, anti-Iba1 for microglia and intrinsic fluorescence or anti-GFP for reporter expression and imaged on a Fluoview 1000 confocal microscope.
Results :
Viral expression was within one 40x field of view at 3-days and multiple 40x fields of view at 4-weeks post injection, with vascular abnormalities observed by 8-weeks post-injection. Localized leaks of fluorescein dye directly correlated to HBs with AAV6-Cre, and leaks or hemorrhage with AAV2-Cre, AAV9-Cre and control eyes were observed by FA. AAV2-Cre induced large, peripheral, singular HBs in approximately 30% of retinas (n=30) and AAV6-Cre induced multiple smaller and more centrally located HBs in approximately 85% of retinas (n=33), both in all three plexus. By contrast, no vascular abnormalities were observed with AAV9-Cre (n=4) or in control eyes (n=30).
Conclusions :
A reproducible, efficient model of VHL HB formation closely mimicking the human clinical presentation was developed using retinal expression ofAAV6-Cre in VHL floxed mice. The efficiency of HB formation with AAV6 suggests a serotype-specific preferential targeting of critical tumorigenic cells. Furthermore, a simple in vivo FA diagnostic will allow facile selection of affected animals for downstream studies.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.