PLATELET-ACTIVATING FACTOR (PAF) RECEPTOR ANTAGONIST TREATMENT INCREASES PROLIFERATION AND PROGENITOR MARKERS EXPRESSION IN RETINAL STEM CELLS

Abstract

<h3>Background</h3> Retinal degenerative diseases are a worldwide public health problem and the main cause of visual loss in humans. Considering stem cell-based therapy as a possible medical approach, quiescent retinal stem cells were identified in the Ciliary Epithelium (CE) of adult mammals; however, regulatory mechanisms inhibit CE stemness, limiting its regenerative potential. It is known that PAF receptor (PAFR) activation suppresses retinal progenitor cell proliferation, suggesting that PAF/PAFR signaling regulates eye stem cells and development. Here we investigated the expression of PAF regulatory enzymes in CE-derived stem cells and the effect of PAFR modulation on proliferation, progenitor, and epithelial markers expression. <h3>Methods</h3> Pigmented epithelium was dissected from mice CE tissue, cultured in DMEN-F12 with 10% FBS for 30 days. Later we add growth factors (EGF/FGF2) to form neurospheres. We analyzed CE and neurospheres transcriptional expression of Pafr, Lpcat1, Pafah1 by real-time PCR. PAFR was modulated during neurosphere forming assay with cPAF (agonist) or PCA4248 (antagonist), and epithelial markers (E-cadherin, N-cadherin), proliferation (Ki67), and progenitor (Pax6 and Nestin) transcripts were analyzed by real-time PCR. An immunohistochemistry assay was performed to investigate proliferation and progenitor markers expression (Ki67 and Sox2). <h3>Results</h3> CE cells downregulated PAF-related enzymes and PAFR expression during neurospheres reprogramming. PAFR agonist inhibited neurosphere formation and controlled epithelial transcriptional expression and morphological phenotype. Oppositely, PAFR antagonist upregulated progenitor transcripts, enhancing CE stemness through increased Ki67, Sox2, Nestin, and Pax6 expression. <h3>Conclusion</h3> Together, our data strongly suggest that PAFR controls CE stemness and can be a promising target for retinal regenerative approaches. <h3>Financial support</h3> FAPESP grant number 2015/24001-1 and 2019/08544-6.