Author: Lucia Gonzalez-Buendia (Spain)
Co-authors: Lucia Gonzalez-Buendia, Santiago Delgado-Tirado, Jose M. Ruiz-Moreno, Joseph F. Alboleda-Velasquez, Leo A. Kim
Purpose
To evaluate the inhibition of Runt-related transcription factor 1 (RUNX1) as a novel therapeutic strategy for choroidal neovascularization (CNV). RUNX1 has been identified as a key mediator of retinal neovascularization by transcriptomic analysis of surgically removed fibrovascular membranes from patients with proliferative diabetic retinopathy. Additionally, RUNX1 inhibition with small-molecule inhibitor Ro5-3335 prevented the formation of retinal neovessels in experimental oxygen-induced retinopathy, a model of retinopathy of prematurity. Furthermore, Ro5-3335 topically administered has recently proved to reduce the severity of proliferative vitreoretinopathy in a rabbit model. CNV is a prevalent cause of blindness in different ocular diseases such as age-related macular degeneration and high myopia. Current treatment for CNV involves repeated intravitreal injections with drugs targeted against vascular endothelial growth factor (VEGF). Despite the initial improvement on visual acuity, several studies show visual decline in the long term, and incomplete response to anti-VEGF agents has been reported in almost half of the patients. Based on our data, we believe that RUNX1 has a critical role on pathologic ocular angiogenesis and that RUNX1 regulation could be a new therapeutic approach to control neovascularization
Setting/Venue
Schepens Eye Research Institute of Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA
Methods
Laser-induced CNV model was used in this study. To evaluate the efficacy of RUNX1 inhibition, a single intravitreal injection of either phosphate-buffered saline (vehicle), Ro5-3335 (75 µmol/L), aflibercept (10 µg) or combination of Ro5-3335 (75 µmol/L)+ aflibercept (10 µg) was performed immediately after laser. Vascular permeability was assessed by fluorescein angiography (FA) performed at day 6 and eyes were enucleated on day 7. RUNX1 expression in CNV lesions was evaluated by immunofluorescence of cryosections and timecourse characterization of its expression was performed by polymerase chain reaction (PCR). All measurements were performed by two different observers in masked and randomized images. CNV lesion size was quantified from images acquired from choroidal flatmounts stained with isolectin B4 using Image-J with Versatile Wand tool plugin. Vascular permeability or leakage area was quantified by measuring the difference between the area of the lesion in the late phase and the early phase of the FA. Finally, leakage severity was assessed by FA image grading. Data were assessed with analysis of variance (one-way ANOVA) followed by Dunnett’s multiple comparisons test. Two-tailed unpaired T-test was used for comparison between two groups
Results
RUNX1 expression was found in the main cell types known to be involved in CNV complex: endothelial cells (CD31+), mononuclear phagocytes (i.e. macrophages/microglia) (CD11b+), retinal pigment epithelial cells (RPE65+), vascular smooth muscle cells/myofibroblasts (alpha-smooth muscle actin+) and Müller cells (glial fibrillary acidic protein+). Importantly, there was no RUNX1 expression in uninjured retina and RUNX1 positive cells were only seen at the lesion site. Fluorescence quantification of choroidal flatmounts at different time points revealed the peak of RUNX1 expression levels on day 3 after laser. mRNA levels quantified by PCR were also higher at day 3. Treatment with Ro5-3335 achieved a significant reduction in CNV area compared with vehicle, but these results were also found in aflibercept and Ro5-3335 + aflibercept groups, in which we saw significantly smaller lesions. Moreover, animals treated with Ro5-3335 and aflibercept exhibited a significant reduction on vascular leakage, but combination of both Ro5-3335 + aflibercept reduced vascular leakage area and severity more effectively than aflibercept or Ro5-3335 alone
Conlusions
In this study, we demonstrate RUNX1 expression in the main cell types involved in a laser-induced model of CNV in mice. Additionally, we report the preclinical efficacy of Ro5-3335, a small-molecule inhibitor of RUNX1, in experimental CNV. Intravitreal injection of Ro5-3335 achieved a significant reduction of CNV area seven days after laser injury, and when combined with aflibercept reduced vascular leakage more effectively than aflibercept in monotherapy. These data suggest that RUNX1 inhibition alone or in combination with anti-VEGF drugs should be further investigated as a novel treatment for patients suffering from CNV
Financial Disclosure
Leo A. Kim and Joseph F. Arboleda-Velasquez: listed as inventors in a patent application for the use of RUNX1 inhibition in aberrant angiogenesis. Jose M. Ruiz-Moreno: Grant of the Spanish Ministry of Health, Instituto deSaludCarlos III, Red Temática de Investigación Cooperativa en Salud: “Prevención, detección precoz, y tratamiento de la patología ocular prevalente, degenerativa y crónica” (RD16/0008/0021). Research support from Topcon, Co. Advisory board of Allergan, Bayer and Novartis. The rest of the authors have no financial relations to disclose.
Comments
-
