Enhancement of NAD–Induced Vasotoxicity: An Early Effect of Diabetes on Retinal Microvessels

Abstract

Purpose: : A sight–threatening complication of diabetes is apoptotic cell death in retinal capillaries. In this study, we tested the hypothesis that extracellular NAD⁺ (nicotinamide adenine dinucleotide) can induce cell death in pericyte–containing retinal microvessels. NAD⁺ is of interest because it is a putative extracellular signaling molecule in the retina and, from studies of immune cells, is reported to interact with P2X₇ purinoceptors whose activation in retinal microvessels can trigger apoptosis.

Methods: : Complexes of pericyte–containing microvessels were freshly isolated from papain–treated adult rat retinas. Cell viability was quantified by trypan blue dye exclusion. Diabetes was induced by streptozotocin–injection.

Results: : In pericyte–containing retinal microvessels, extracellular NAD⁺ caused cell death in a dose–dependent manner (EC₅₀ = 2 nM). Of importance to diabetes, the NAD⁺ concentration needed to cause microvascular cell death decreased by ∼40–fold soon after the onset of streptozotocin–induced hyperglycemia. Consistent with NAD’s vasotoxicity being mediated via P2X₇ receptor activation, two blockers of these purinoceptors, oxidized–ATP and Brilliant Blue G, dramatically reduced (P = 0.001) the microvascular death caused by NAD⁺. In addition, cell death induced by NAD⁺ was prevented (P < 0.001) by pre–exposure to UTP, which in retinal microvessels activates P2Y receptors and blocks the creation of pores formed by P2X₇ activation. Indicative of the importance of NAD⁺ rather than its catabolites, neither ADP–ribose nor nicotinamide were vasotoxic. Suggestive that ecto–ADP–ribosyltransferases (ecto–ARTs) play a key role, pre–incubation of microvessels with a relatively high concentration of etheno–NAD⁺ (100 µM), which is an ART substrate that does not cause microvascular death, prevented (P = 0.001) NAD⁺ from inducing vasotoxicity.

Conclusions: : Our findings support the hypothesis that extracellular NAD⁺ can kill retinal microvascular cells by a mechanism involving the activation of P2X₇ purinoceptors. Of further importance, the vulnerability of pericyte–containing retinal microvessels to NAD–induced vasotoxicity was dramatically increased soon after the onset of experimental diabetes.

Keywords: diabetic retinopathy • apoptosis/cell death • vascular cells