PURPOSE: Retinal hypoxia-mediated activation of the hypoxia-inducible factor (HIF pathway) leading to angiogenesis is a major signaling mechanism underlying a number of sight-threatening diseases. Inhibiting this signaling mechanism with an already approved therapeutic molecule may have promising anti-angiogenic role with fewer side effects. Hence, the primary objective of this study was to examine the expression of HIF-1α and VEGF in human retinal pigment epithelial cells treated with ritonavir under hypoxic and normoxic conditions. METHODS: ARPE-19 and D407 cells were cultured in normoxic or hypoxic conditions, alone or in the presence of ritonavir. Quantitative real-time polymerase chain reaction, immunoblot analysis, sandwich ELISA, endothelial cell proliferation, and cytotoxicity were performed. RESULTS: A 12-h hypoxic exposure resulted in elevated mRNA expression levels of both HIF-1α and VEGF in ARPE-19 and D407 cells. Hence, this time point was selected for subsequent experiments. Presence of ritonavir in the culture medium strongly inhibited VEGF expression in a concentration-dependent manner under hypoxic conditions. Immunoblot analysis demonstrated a substantially reduced protein expression of HIF-1α in the presence of ritonavir. Further, hypoxic exposure-induced VEGF secretion was also inhibited by ritonavir, as demonstrated using ELISA. Finally, ritonavir significantly diminished the proliferation of choroid-retinal endothelial (RF/6A) cells demonstrating potential anti-angiogenic activity. Cytotoxicity studies showed that ritonavir is non-toxic to RPE cells. CONCLUSIONS: This study demonstrates for the first time that ritonavir can inhibit HIF-1α and VEGF in ARPE-19 and D407 cells. Such inhibition may form a platform for application of ritonavir in the treatment of various ocular diseases.
PURPOSE: Retinal hypoxia-mediated activation of the hypoxia-inducible factor (HIF pathway) leading to angiogenesis is a major signaling mechanism underlying a number of sight-threatening diseases. Inhibiting this signaling mechanism with an already approved therapeutic molecule may have promising anti-angiogenic role with fewer side effects. Hence, the primary objective of this study was to examine the expression of HIF-1α and VEGF in human retinal pigment epithelial cells treated with ritonavir under hypoxic and normoxic conditions. METHODS:ARPE-19 and D407 cells were cultured in normoxic or hypoxic conditions, alone or in the presence of ritonavir. Quantitative real-time polymerase chain reaction, immunoblot analysis, sandwich ELISA, endothelial cell proliferation, and cytotoxicity were performed. RESULTS: A 12-h hypoxic exposure resulted in elevated mRNA expression levels of both HIF-1α and VEGF in ARPE-19 and D407 cells. Hence, this time point was selected for subsequent experiments. Presence of ritonavir in the culture medium strongly inhibited VEGF expression in a concentration-dependent manner under hypoxic conditions. Immunoblot analysis demonstrated a substantially reduced protein expression of HIF-1α in the presence of ritonavir. Further, hypoxic exposure-induced VEGF secretion was also inhibited by ritonavir, as demonstrated using ELISA. Finally, ritonavir significantly diminished the proliferation of choroid-retinal endothelial (RF/6A) cells demonstrating potential anti-angiogenic activity. Cytotoxicity studies showed that ritonavir is non-toxic to RPE cells. CONCLUSIONS: This study demonstrates for the first time that ritonavir can inhibit HIF-1α and VEGF in ARPE-19 and D407 cells. Such inhibition may form a platform for application of ritonavir in the treatment of various ocular diseases.
Authors: Eugene W M Ng; David T Shima; Perry Calias; Emmett T Cunningham; David R Guyer; Anthony P Adamis Journal: Nat Rev Drug Discov Date: 2006-02 Impact factor: 84.694
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Authors: Anyan Xie; René J Robles; Samiran Mukherjee; Haohai Zhang; Linda Feldbrügge; Eva Csizmadia; Yan Wu; Keiichi Enjyoji; Alan C Moss; Leo E Otterbein; Francisco J Quintana; Simon C Robson; Maria Serena Longhi Journal: J Autoimmun Date: 2018-08-08 Impact factor: 7.094