Abstract
Purpose :
Choroidal neovascularization (CNV) describes the growth of abnormal blood vessels from the choroid into the sub-retinal pigment epithelium or subretinal space and is typically accompanied by severe vision loss. CNV is a common complication in Sorsby fundus dystrophy (SFD), a rare autosomal dominant maculopathy caused by mutations in the TIMP3 gene. Here we performed ex vivo choroid angiogenesis assays to evaluate vascular sprouting in wild-type mice and different TIMP3-gene targeted mouse models.
Methods :
Ex vivo choroid sprouting assays using RPE/choroid/sclera-tissue punches of wild-type mice, TIMP3-deficient (knock-out) mice and mice carrying the SFD-related TIMP3 mutation p.S156C (knock-in) (CD1 background) at various ages (4 weeks, 3 and 6 months) were performed. Standardized quantification of phase contrast photos of the sprouts were done with ImageJ. Sprouting area (mm2), growth rate and onset of sprout growth were determined and the data were statistically evaluated.
Results :
Images of sprouts from a total of 1011 tissue punches from 145 mice were analyzed and quantified. In general, sprouting from choroidal tissue was significantly faster in 4-week old compared to the older mice. A significant difference in growth rate and onset of sprouting was observed between wild-type and TIMP3 knock-in mice; the presence of the p.S156C mutation clearly promoted choroidal sprouting (TIMP3S156C/S156C > TIMP3S156C/+). This difference was most pronounced for the 4-week old animals. In addition, explants from young TIMP3 knock-in mice also showed a significant larger vessel sprouting area than explants from wild-type mice. In contrast, comparison of the sprouting area from choroidal punches of wild-type and TIMP3-deficient mice revealed that loss of TIMP3 significantly suppressed vessel sprouting. The slowing effect on vascular angiogenesis mediated by TIMP3 deficiency, however, could not be confirmed when comparing growth rate and growth onset of choroidal sprouting in wild-type and TIMP3 knock-out explants.
Conclusions :
Here we show that mutated p.S156C-TIMP3 but not TIMP3 deficiency results in a promoting effect on microvessel sprouting in our choroid angiogenesis assay. Importantly, this assay can be used to test the influence of pharmacologic substances on the formation and prevention of angiogenesis and may therefore be most efficient to identify drugs for treatment of CNV in SFD and related diseases.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.