Purchase this article with an account.
Ramya Krishna Vadlapatla, Aswani Dutt Vadlapudi, Dhananjay Pal, Mridul Mukherji, Ashim Mitra; Ritonavir Inhibits HIF-1α Mediated VEGF Expression in Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3275.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Retinal hypoxia (oxygen deficiency) leading to angiogenesis is a major signaling mechanism underlying a number of sight threatening diseases. One of the most important target genes of hypoxia inducible factor (HIF) includes vascular endothelial growth factor (VEGF). Intravitreal injections of VEGF antagonists resulted in serious complications and systemic side effects. Inhibiting the signaling mechanism underlying neovascularization (HIF-pathway) with already approved therapeutic molecule may have promising anti-angiogenic role with fewer side-effects. Hence, the primary objective of this study is to examine the expression of HIF-1α and VEGF in human retinal pigment epithelial (ARPE-19 and D407) cells treated with ritonavir under hypoxic and normoxic conditions.
ARPE-19 and D407 cells were cultured in normoxic and hypoxic conditions (3, 6 and 12 hours), alone or in the presence of ritonavir (5, 10 and 20μM). Quantitative real time polymerase chain reaction (qPCR), immunoblot analysis and sandwich ELISA were performed to check the RNA and protein expression levels of HIF-1α and VEGF. Further, in vitro anti-angiogenic activity of ritonavir was also assessed by measuring proliferation of choroid-retinal endothelial (RF/6A) cells.
Hypoxic exposure to 12 hours demonstrated elevated RNA expression levels of HIF-1α and VEGF in both ARPE-19 and D407 cells. Hence, this time point was chosen for subsequent studies. Presence of ritonavir in culture medium strongly inhibited the expression of HIF-1α and VEGF in a concentration-dependent manner in hypoxic conditions. Immunoblot analysis demonstrated that ritonavir inhibited the protein expression of HIF-1α. Further, hypoxic exposure increased VEGF secretion while presence of ritonavir reduced this secretion, as demonstrated by an ELISA. Finally, ritonavir significantly inhibited the proliferation of RF/6A cells, demonstrating potential anti-angiogenic activity.
This study demonstrates for the first time that ritonavir can inhibit the expression of HIF-1α and VEGF in ARPE-19 and D407 cells. This inhibition of hypoxia pathway and downstream genes may form the platform for indication of ritonavir in treatment of various sight threatening diseases. The process of traditional drug development could be fastened since ritonavir is clinically approved for human use.
This PDF is available to Subscribers Only