title: An in vitro retinoblastoma human triple culture model of angiogenesis: a modulatory effect of TGF-β.
abstract: Retinoblastoma is the most common intraocular tumour in children. In view of understanding the molecular mechanisms through which angiogenic switch on happens in the early phases of reciprocal interaction between tumour and cells constituting retinal microvessel, Transwell co-cultures constituted by human retinal endothelial cells (HREC), pericytes (HRPC), and human retinoblastoma cell line Y-79 were performed. Y-79 enhanced HREC proliferation, reduced by the introduction of HRPC in triple culture. In HREC/HRPC cultures, TGF-β in media increased, decreasing in triple cultures. High VEGF levels in triple cultures witnessed the establishment of a strongly in vitro angiogenic environment. Y-79 induced in HREC an increase in c- and iPLA2, phospho-cPLA2, inducible COX-2 protein expressions, PLA2 activities and prostaglandin E2 (PGE2) release. These effects were attenuated when HRPC were introduced in triple culture. Moreover, antibody silencing of TGF-β demonstrated a strong correlation between the signalling pathway triggered by TGF-β of pericytal origin and the phospholipase activation and the modulation of PGE2 release. Inhibiting VEGFA effect, the HRPC loss in triple culture decreased, showing its modulatory effect on their survival. Relying on the data here presented, sustaining the pericytal survival in a tumour retinal environment could ensure the integrity of microvessels and the TGF-β supply, essential for controlling aberrant endothelial pruning and angiogenesis.
authors.affiliation: Dipartimento di Biomedicina Clinica e Molecolare, Università di Catania, Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Lupo
authors.affiliation: Dipartimento di Biomedicina Clinica e Molecolare, Università di Catania, Italy.
authors.forename: Carla
authors.initials: C
authors.lastname: Motta
authors.affiliation: Dipartimento di Scienze Bio-Mediche, Università di Catania, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Salmeri
authors.affiliation: Dipartimento di Scienze Bio-Mediche, Università di Catania, Italy.
authors.forename: Vittoria
authors.initials: V
authors.lastname: Spina-Purrello
authors.affiliation: Dipartimento di Biomedicina Clinica e Molecolare, Università di Catania, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Alberghina
authors.affiliation: Dipartimento di Biomedicina Clinica e Molecolare, Università di Catania, Italy. Electronic address: anfudan@unict.it.
authors.forename: Carmelina Daniela
authors.initials: CD
authors.lastname: Anfuso
mesh_terms: D049109:Cell Proliferation; D002470:Cell Survival; D018920:Coculture Techniques; D003600:Cytosol / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008833:Microcirculation; D009389:Neovascularization, Pathologic; D010539:Permeability; D010740:Phospholipases / Q000378:metabolism; D054467:Phospholipases A2 / Q000378:metabolism; D012160:Retina / Q000378:metabolism; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D016212:Transforming Growth Factor beta; D010740:Phospholipases; D054467:Phospholipases A2
keywords: Cytosolic and calcium-independent PLA(2); Human retinal microvascular endothelial cells; Human retinal microvascular pericytes; Retinoblastoma; TGF-β