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T.E. Walshe, C. O'Brien, P.A. Cahill; Notch Signaling in Co–Cultured Endothelial & Pericyte Cells Exposed to Pulsatile Flow . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4654.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The Notch signaling pathway is a highly conserved mechanism of cell–to–cell communication involved in cell fate decisions. Ligand activation of the Notch receptor on the cytoplasmic membrane releases the intracellular portion which localises to the nucleus and causes transcription of target genes that regulate apoptosis & proliferation. Abnormalities in retinal blood flow are a precursor of remodeling of the retinal vasculature, which alter apoptosis, proliferation and migration of vascular cells.Using a novel perfused transcapillary hemodynamic co–culture of bovine retinal endothelial cells (BRECs) & bovine retinal pericytes (BRPs), we examined the proliferative and apoptotic profile of each cell type and the role of the Notch signaling pathway. Methods:Using a perfused transcapillary culture system, co–cultures of BRECs with BRPs were exposed to low and high pulsatile flow for 72 hours (corresponding to a low flow rate of 0.3 mls/min; 6 mmHg pulse pressure; 0.5 dyn/cm2 shear stress and a high flow rate of 24 mls/min, 56 mmHg pulse pressure; 23 dyn/cm2 shear stress). Each cell type were harvested separately and analysed via flow cytometry, real time PCR and western blotting. Results: FACs analysis of BRP demonstrated increased apoptosis and decreased proliferation when exposed to high pulsatile flow. BRP pCNA protein also decreased. Pro–apoptotic bax mRNA and protein increased in BRP exposed to high pulsatile flow, whereas the anti–apoptotic marker BCl–2, but not BCl–xL, decreased as determined by RT–PCR and western blotting. High flow reduced BRP Notch–1 mRNA and receptor cleavage, whereas no change in Notch–3 receptor cleavage was found. In contrast, high pulsatile flow reduced BREC apoptosis, whereas proliferation was unchanged after 72 hours. Also, BREC pCNA protein was unchanged. High flow decreased BREC Bax mRNA and protein, whereas anti–apoptotic markers BCl–2 and BCl–xl mRNA and protein were increased. Notch–1 mRNA and receptor cleavage increased under high flow. The anti–apoptotic effect of high flow is reversed in BRECs transfected with RPMS–1 which binds to Notch–1, preventing downstream signalling. Conclusions: These results demonstrate Notch–1 receptors are expressed in retinal microvascular cells and that pulsatile flow modulates their expression in both BRPs and BRECs in co–culture. Furthermore, pulsatile flow has a pro–apoptotic effect in BRPs and an anti–apoptotic effect in BRECs, possibly mediated via Notch–1 signalling.
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