3,3′-Diindolylmethane inhibits VEGF expression through the HIF-1α and NF-κB pathways in human retinal pigment epithelial cells under chemical hypoxic conditions

Abstract

Oxidative stress in the retinal pigment epithelium (RPE) can lead to the pathological causes of age-related macular degeneration (AMD). Hypoxia induces oxidative damage in retinal pigment epithelial cells (RPE cells). In this study, we investigated the capacity of 3,3'-diindolylmethane (DIM) to reduce the expression of vascular endothelial growth factor (VEGF) under hypoxic conditions, as well as the molecular mechanisms involved. Human RPE cells (ARPE-19 cells) were treated with cobalt chloride (CoCl2, 200 µM) and/or DIM (10 and 20 µM). The production of VEGF was measured by enzyme-linked immunosorbent assay. The translocation of hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-κB (NF-κB) was determined by western blot analysis. The binding activity of HIF-1α and NF-κB was analyzed by electrophoretic mobility shift assay. The phosphorylation levels of mitogen-activated protein kinases (MAPKs) were measured by western blot analysis. The levels of mitochondrial reactive oxygen species (ROS) were detected by fluorescence microplate assay. The results revealed that DIM significantly attenuated the CoCl2-induced expression of VEGF in the ARPE-19 cells. The CoCl2-induced translocation and activation of HIF-1α and NF-κB were also attenuated by treatment with DIM. In addition, DIM inhibited the CoCl2-induced activation of p38 MAPK in the ARPE-19 cells. Pre-treatment with YCG063, a mitochondrial ROS inhibitor, led to the downregulation of the CoCl2-induced production of VEGF by suppressing HIF-1α and NF-κB activity. Taken together, the findings of our study demonstrate that DIM inhibits the CoCl2-induced production of VEGF by suppressing mitochondrial ROS production, thus attenuating the activation of HIF-1α and p38 MAPK/NF-κB.

Microfluidic co-cultures of retinal pigment epithelial cells and vascular endothelial cells to investigate choroidal angiogenesis

“…Increasing non-hexagonal RPE cell shapes, reminiscent of the SIPS characteristic alterations of cell shape, has been described in aging retinas and AMD donor eyes 26 . We resorted to using CoCl 2 to mimic hypoxia due to the limited numbers of incubators available and also considered the fact that the cobalt chloride method is not only an inexpensive way in inducing HIF-1 alpha production, but also avoids oxygen re-entering the chamber every time images were taken which might affect the results 27, 28 . We finalized the concentration of CoCl 2 to be 150 µM, the non-lethal hypoxic concentration after determining viability of ARPE-19 cells under CoCl 2 of 100–300 µM (data not shown).…”

Effects of VEGF inhibitors on human retinal pigment epithelium under high glucose and hypoxia

“…Hypoxia was induced chemically through the addition of cobalt chloride (CoCl 2 ; Sigma, St. Louis, Missouri). CoCl 2 mimics the hypoxic state through induction of hypoxia inducible factor 1‐alpha (HIF‐1α) activity in many mammalian cell lines including ARPE‐19 . HIF‐1a is considered the master regulator of hypoxia and mediates homeostatic response to low oxygen states …”

Mitochondrial ROS control of cancer

“…In addition to cellular proliferation, angiogenesis is induced by the upregulation of vascular endothelial growth factor (VEGF), by mROS transcriptionally and through mROS-induced HIF stabilization 40–42 . mROS-induced HIF stabilization allows for its binding to hypoxia response element (HRE) to express hypoxic adaptation genes 43,44 in an adaptive response to low oxygen levels.…”