Abstract
Purpose :
Accelerated retinal endothelial cell senescence and acquisition of a senescence-associated secretory phenotype is an important pathogenic mechanism contributing to induction and progression of diabetic retinopathy (DR). To better understand the molecular basis of this mechanism, we have investigated the specific contribution of NAD+ and nicotinamide phosphoribosyltransferase (NAMPT), the enzyme responsible for NAD+ production, in human and experimental (DR) as well as in human retinal endothelial cells exposed to glucidic stress.
Methods :
Postmortem human diabetic and non-diabetic donors were used to assess NAD+ and NAMPT levels in human retinas. In vivo studies were conducted using streptozotocin-induced diabetic rats at 8 weeks of hyperglycemia. In vitro studies were conducted in human retinal endothelial cells (HuREC) exposed to glucidic stress (25mM D-glucose =HG) or to control conditions (25mM L-glucose =LG; 5mM D-glucose=NG). NAD+ levels were assessed using a commercially available kit. NAMPT expression was measured by qPCR and Western blotting. In vitro inhibition of NAMPT was achieved using the specific inhibitor FK866 at different doses. SASP was demonstrated in HuREC by assessing senescensce-associated beta-galactosidase activity (SA-beta-gal) and expression of p16INK4a, p21, interleukin 1beta (IL-1beta) and sirtuin-1 (SIRT-1).
Results :
NAD+ content was significantly down-regulated in human DR and STZ-rat retinas. Loss of NAD+ correlated with significant decrease in protein and mRNA levels of the NAMPT enzyme. HuREC exposed to HG and not to the controls (LG or NG) demonstrated SASP characteristics such as increased SA-beta-gal activity and increased expression of senescence markers (p16INK4a, p21 and IL-1beta) and loss of SIRT-1. This effect was associated with significant decrease of NAMPT at protein and mRNA levels. Inhibition of NAMPT activity by the specific inhibitor FK866 produced a progressive dose-dependent decrease in NAD+ and SIRT-1 and increase in SASP-related factors in HuREC.
Conclusions :
Our studies have identified in dysfunction of NAD+/NAMPT levels and related function a critical new pathogenic mechanism involved in hyperglycemia –induced retinal microvascular senescence and DR progression.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.