KRN-633

Vascular endothelial growth factor (VEGF) promotes physiological and pathological retinal angiogenesis by activating the mammalian target of rapamycin complex 1 (mTORC1) pathway in proliferating endothelial cells. This study aimed to visualize the status of the VEGF receptor (VEGFR) pathway in endothelial cells by assessing the phosphorylation of S6 protein (pS6), a downstream marker of mTORC1 activity, in the neonatal mouse retina. Four-day-old mice received subcutaneous injections of rapamycin (mTORC1 inhibitor), PF-4708671 (S6 kinase 1 inhibitor), KRN633 (VEGFR tyrosine kinase inhibitor), or vehicle. The eyes were collected 1, 3, 6, 24, and 48 h after treatment. The vascular density, pS6 distribution, and proliferative activity were evaluated in the retina. pS6 immunoreactivity was detected in developing blood vessels, astrocytes, and microglial cells. Both rapamycin and PF-4708671 almost completely abolished pS6 immunoreactivity in vascular and non-vascular cells 6 h after treatment and thereafter suppressed endothelial cell proliferation before the onset of capillary degeneration. In contrast, KRN633 markedly reduced pS6 immunoreactivity associated with endothelial cells, but not in non-vascular cells at 6 h post-treatment; however, capillary endothelial cell degeneration became apparent at 24 h. These results suggest that VEGFR inhibition disrupts the mTORC1 pathway in endothelial cells within 6 h of treatment, causing endothelial cell degeneration or death in developing retinal blood vessels. Monitoring changes in pS6 immunoreactivity before the onset of endothelial cell degeneration may serve as a valuable method for assessing endothelial responses to VEGF in the retina.

Retinopathy of prematurity (ROP), caused by oxidative stress leading to abnormal retinal vessel growth, is often studied in rodent models. However, current methods rely on in vitro analysis, preventing the monitoring of disease progression. Fundus imaging is commonly used in clinical ophthalmology and could provide a method for in vivo imaging in rodents with ROP. Recently, a smartphone and condensing lens have been shown to be effective in humans and mice. The aim of this study was to develop a smartphone-based fundus camera system that can be used to monitor in vivo changes caused by ROP and determine its effectiveness upon introducing an intervention of lutein. KRN 633 was used to induce ROP and fundus imaging was conducted using a condensing lens and a smartphone. In vitro analysis of the retina was conducted for comparison using immunohistochemistry. The tortuosity was analyzed from fundus images manually using ImageJ and by a newly developed computer vision-based method. ROP induced tortuous arteries, which was improved by the administration of lutein in the fundus and microscopy images of the retina. The two imaging techniques showed a strong positive correlation. Similarly, the computer vision-based tortuosity analysis displayed a strong linear relationship with ImageJ analysis. This smartphone-based fundus camera system offers a cheap and accessible method for in vivo imaging to supplement traditional histological analysis. With future development, the image processing technique described can be enhanced to include other measures of visual function.