Neuroprotective effects of PACAP, VIP and NAP against hyperglycaemic retinal damage

Abstract

Diabetic retinopathy, one the most significant and disabling chronic complications of diabetes mellitus, has mainly been regarded as a microcirculatory disease of the retina. However, there is emerging evidence to suggest that a primary and early neurodegenerative process characterizes DR. The reason for the retinal degeneration resides in the metabolic changes caused by hyperglycemia and in the decreased capability of the retina to adapt to this environment. This situation leads to altered expression patterns of neuropeptides, growth and transcription factors, apoptosis, increased oxidative stress, inflammatory response, angiogenesis and disruption of the blood-retinal barrier (BRB). All major cell types of the retina are affected: neuronal as well as the glial cells and pigment epithelial cells. Pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and davunetide (NAP) are three pleiotropic related neuropeptides also expressed in the retina. Their protective role has been shown in different retinal injuries, but little is known about the relationship between these peptides and DR. The aim of the present thesis was to investigate, using both in vitro and in vivo model of DR, the role of these peptdides on retinal layers by monitoring apoptotic events by western blot and confocal microscopy analysis and measuring the integrity of the outer blood retinal barrier with permeability and transepithelial electrical resistance assay. The underlying signal transduction pathways activated by each peptide and the impact of hyperglicaemia on the expression and distribution at retinal levels of the inflammatory IL-1 mediators were also evaluated. Diabetes was mimicked in adult rats by intraperitoneal injection of streptozotocin (STZ) and human retinal pigment epithelial cells (ARPE19) were cultured for 26 days in high glucose (25mM of D-glucose) and IL-1beta. The results confirmed that hyperglycemia induced early apoptotic death in the cellular components of the neuroretina, breakdown of the outer blood retinal barrier (BRB) and dysregulation of several components of the metabolic and signaling pathways. The neuropeptides tested activate promising pathways useful for the treatment of this retinal degenerative disease. In particular, PACAP and VIP promote the integrity of the outer BRB, possibly through the modulation of proteins related to tight junctions. Davunetive is able to reduce apoptosis in the diabetic retina by activating the anti-apoptotic p-Akt, p-ERK1, p-ERK2, Bcl-2 and decreasing levels of the pro-apoptotic elements, such as cleaved caspase-3. These results raise the opportunity for the use of these peptides as a possible therapeutic or preventive methods in treating diabetes.