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Temitope Sasore, Roisin Dunne, Breandan N Kennedy; Can drug combinations targeting PI3K/Akt/mTOR pathway inhibit angiogenesis, inflammation or permeability in human RPE and endothelial cell lines?. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2305.
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© ARVO (1962-2015); The Authors (2016-present)
Age-related macular degeneration (AMD), diabetic retinopathy (DR) and diabetic macula edema (DME) are characterised by inflammation, retinal angiogenesis and increased retinal vascular permeability (RVP). There is an urgent need to develop better therapies to treat these diseases. Previously, we showed that combinations of PI3K/Akt/mTOR (PAM) pathway inhibitors have additive or synergistic anti-angiogeneic activity in vivo (Sasore and Kennedy 2014). Here, we assess the anti-angiogenic, anti-inflammatory or anti-RVP of these drugs in human RPE and endothelial cells.
Expression of PAM target genes is confirmed in human cell lines by reverse transcription polymerase chain reaction (RTPCR). Western blot using phospho-specific antibodies assessed activation of PAM pathway signalling. The secretion of angiogenic and inflammatory factors was assessed using multiplex ELISA. Localization of zonula occludens-1 (ZO-1) tight junction protein is used to evaluate barrier integrity by immunocytochemistry.
PAM pathway target genes (PIK3CA, PIK3R1, AKT1, MTOR, RPS6KB1 and EIF4EBP1) are expressed in human ARPE-19. The most active drug combinations additively inhibit phosphorylation of p70S6K protein in human RPE cells. Preliminary data indicate that individual PAM pathway drugs reduce sVCAM-1 expression, with drug combinations having stronger effects. These drugs also reverse barrier disruption induced in ARPE-19 cells as determined by localization of ZO-1.
In summary, our study identifies combinations of PI3K/Akt/mTOR pathway inhibitors that are effective inhibitors of angiogenesis and cellular permeability in vitro. Further studies will evaluate the effect of these drugs on inflammation and permeability using multiplex ELISA and trans-epithelial/endothelial electrical resistance (TEER), respectively. In conclusion, the combination of PI3K/Akt/mTOR pathway drugs hold promise as inhibitors of retinal angiogenesis, inflammation and retinal vascular permeability.
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