Exploring the SLIT-ROBO Pathway in the Pathogenesis of Sickle Cell Retinopathy: In Vitro Analysis using ARPE-19 Retinal Cells and Patients Serum

Abstract

The term sickle cell disease (SCD) is used to characterize a group of hemoglobinopathies that have a point mutation in the seventh codon of the beta-globin gene in common, located on chromosome 11p15.5.7. Ophthalmological complications are common in sickle cell diseases, including conjunctiva abnormalities, orbital infarctions, retinopathy, and retinal hemorrhage. Retinal alterations are the most important for ocular morbidity in sickle cell diseases, especially the proliferative form, which is the major cause of progressive vision loss in these patients, leading to visual impairment in 10 to 20% of affected eyes. The estimated prevalence of proliferative sickle cell retinopathy (PSCR) is 32.8% in HbSC compound heterozygotes, 14% in S-beta thalassemia, and 2.6% in sickle cell anemia (HbSS homozygotes).The pathophysiology of SCR relies on the aggregation of sickled red blood cells, activation of the endothelium and thromboinflammatory cascades, which culminate in arteriolar occlusion of the peripheral retina. In PSCR, the resulting ischemia stimulates neovascularization with the formation of fragile vessels, which can lead to vitreous hemorrhage and retinal detachment, as vision loss. The precise cause of neovessel formation in sickle cell diseases is not fully understood, so retinopathy in these individuals offers an intriguing model for study, given its link with a systemically milder genotype, BSBC, and the common property of spontaneously regressing.The SLIT-ROBO pathway has been studied to play an essential role in vasculogenesis and angiogenesis, including ocular angiogenesis. A previous study by our research group used RNA-Seq to evaluate the gene expression profile of endothelial colony-forming cells (ECFCs) from SS and SC patients with and without PSCR (process FAPESP 2015/14255-6). As a result, differentially expressed genes involved in cell proliferation, angiogenesis, and inflammation pathways were found, highlighting the change in gene expression of the Roundabout guidance receptor 1 (ROBO1) gene. Studies have described the participation of ROBO1 in angiogenesis induced by VEGF-A, indicating its importance in angiogenic processes. The ROBO1 gene is expressed in retinal vessels and plays a fundamental role in retinal neovascularization, particularly during superficial and deep vascular plexus formation. Furthermore, SLIT2 has been extensively studied for its involvement in vasculogenesis. Although some studies describe the SLIT-ROBO pathway, its role in sickle cell retinopathy has yet to be demonstrated.Due to limitations in obtaining human retinal samples for experimental studies, the main objective of this project is to conduct in vitro assays to evaluate the role of the SLIT-ROBO signaling pathway in sickle cell retinopathy. Furthermore, we seek to identify targets downstream of this pathway that may be associated with the development of SCR, contributing to a better understanding of the molecular mechanisms involved in the development of RF. This study will use peripheral blood from HbAA, HbSS, and HbSC patients with and without sickle cell retinopathy to treat the ARPE-19 lineage, a retinal pigment epithelium cell line. After treatment, ARPE-19 cells, with and without supplementation, will be subjected to experimental protocols: RNA and protein extraction, cell proliferation/viability assay, invasion and migration assay apoptosis assays, and qRT-PCR and western blot analysis. Therefore, the results obtained in this project may contribute to a better understanding of the mechanisms involved in sickle cell retinopathy and serve as a basis for subsequent studies. (AU)