title: Ritonavir inhibits HIF-1α-mediated VEGF expression in retinal pigment epithelial cells in vitro.
abstract: OBJECTIVE
  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.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO, USA.
authors.forename: R K
authors.initials: RK
authors.lastname: Vadlapatla
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO, USA.
authors.forename: A D
authors.initials: AD
authors.lastname: Vadlapudi
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO, USA.
authors.forename: D
authors.initials: D
authors.lastname: Pal
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO, USA.
authors.forename: M
authors.initials: M
authors.lastname: Mukherji
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO, USA.
authors.forename: A K
authors.initials: AK
authors.lastname: Mitra
mesh_terms: D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D006801:Humans; D000860:Hypoxia / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D019438:Ritonavir / Q000494:pharmacology*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
chemical_list: C497442:HIF1A protein, human; D017320:HIV Protease Inhibitors; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D019438:Ritonavir