Purchase this article with an account.
Harshini Chakravarthy, Todd Lydic, Qi Wang, Svetlana Bozack, Prathiba Jayaguru, Susanne Mohr, Julia Busik; The Role Of Sphingolipid Metabolism In Disruption Of Circadian VEGF Production By Retinal Cells Under Hyperglycemic And Pro-inflammatory Conditions. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4334.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We previously showed that retinal pigment epithelial cells (RPE) and Müller cells represent the major source of VEGF secretion under cytokine and high glucose stimulation. Diabetes is also believed to be associated with a phase shift and dampening of oscillations of peripheral clock genes which points to a disturbed circadian rhythm. In this context, we tested for circadian variations in production of VEGF when synchronized RPE and Müller cells are challenged with cytokines or high glucose concentrations. To elucidate the mechanism of cytokine and high glucose induced changes in circadian VEGF production, the role of sphingomyelinase activation, representing the initial step of cytokine signaling was evaluated.
Primary cultures of RPE and Müller cells were treated with 2 ng/mL IL-1β for 24 hours or 25 mM glucose for upto 96 hours. Sphingomelinases were inhibited by siRNA approach or pretreatment with 15 µM desipramine or 25 µM GW4869. 100 nM Dexamethasone was used to synchronize RPE and Müller cells grown in media containing 25 mM glucose for 24 hours. Induction of ICAM-1 by IL-1β was determined by Western Blot and VEGF production was measured by ELISA. Müller cells were treated with 100 µM Minocycline, a Caspase-1 inhibitor and grown in media containing 25 mM glucose to assess the effect of inhibition of Caspase-1 mediated IL-1β production on high glucose induced sphingomyelinase activation.
In agreement with our previously reported study, high glucose induces a 9.3-fold increase in IL-1β production in RPE cells and 12-fold increase in Müller cells. In turn, IL-1β strongly induces VEGF production starting at 12 hours in Müller cells. After 24 hours, VEGF secretion was increased 3.2-fold relative to control. Minocycline prevented increase in ceramide levels seen in high glucose conditions by 2.9-fold confirming that IL-1β stimulation causes changes in sphingolipid metabolism in Müller cells. VEGF production followed a distinct circadian pattern in RPE and to a lesser extent, in Müller cells. High glucose caused a phase shift in the circadian pattern in both cell types. Interestingly, inhibition of ASM increased IL-1β induced VEGF secretion at 24 hour time point, indicating that ASM is an important regulator of circadian VEGF secretion and IL-1β signaling.
The data obtained by these experiments implicates high glucose induced IL-1β production and sphingomyelinase activation in RPE and Müller cells in retinal VEGF secretion. Disruption of circadian rhythmicity of VEGF production by high glucose and cytokines in RPE and Müller cells could represent an unexploited mechanism in the development of diabetic retinopathy.
This PDF is available to Subscribers Only