New purinergic-based pharmacological targets for the treatment of retinal disorders

Diabetic retinopathy (DR) is a progressive disease representing a major microvascular complication of diabetes and a leading cause of visual loss among adults in most industrialized countries. The prevalence of DR is expected to raise worldwide due to aging population, longer life expectancy, and a higher prevalence of people with diabetes, although risk factors management in diabetes care has been improved. The hyperglycemic environment, typical of DR onset, induces blood retinal barrier (BRB) breakdown along with an enhanced expression of pro-inflammatory cell markers and oxidative stress-related mediators. Additionally, the high glucose (HG) levels observed during diabetes induce the activation of the purinergic signaling pathway, which includes the P2X7 receptor (P2X7R). This ATP-gated ion channel has been linked to vascular inflammation due to an over-expression of cytokines, contributing to BRB alteration. The integrity of this physical barrier is essential for a proper vision; indeed, the BRB dysfunction mainly contributes to the development of DR by leading to vascular leakage to surrounding tissues and the consequent vision impairments. Based on the above, I focused my research project on the development of an innovative strategy for the early treatment of DR through the negative modulation of P2X7R and the decrease of inflammatory cytokines, both of them playing a pivotal role in BRB damage. First of all, I set up a BRB triple co-culture model entirely based on human cells (retinal endothelial cells, retinal pericytes, and retinal astrocytes), in order to mimic the human milieu, characterized by the same cellular numerical ratio and layer order observed in vivo. The results obtained by using this innovate system showed that an exposure for 48 hours to HG provoked the BRB breakdown, increased the barrier permeability (measured by trans-endothelial electrical resistance (TEER)), and reduced the levels of junctional proteins such as vascular endothelial (VE)-cadherin and zonula occludens-1 (ZO-1). In our human inner BRB (iBRB) model, the hyperglycemic environment also led to the over-expression of inflammatory mediators (IL-1β, IL-6) and oxidative stress-related genes (iNOS, Nox2) along with a significant increase in reactive oxygen species (ROS) formation and the activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/heme-oxigenase-1 (HO-1) antioxidant axis. Once the model was established, I aimed to investigate the role played by P2X7R in the observed iBRB damage. I first indentified a novel P2X7R antagonist through a virtual screening analysis on a small in-house compound dataset. The results obtained by the computational analysis revealed that the diterpenoid dihydrotanshinone (DHTS) clustered with the well-known P2X7R antagonist JNJ47965567 (used as a positive P2X7R antagonist control in my experiments). In order to assess the potential protective effect of DHTS (as a possible P2X7R antagonist), I challenged the iBRB model with a combination of HG and 2′(3′)-O-(4-Benzoylbenzoyl)adenosine-5′-triphosphate (BzATP) (a selective P2X7R agonist) in absence or presence of this diterpenoid. I found that HG/BzATP stimulation led to an enhanced barrier permeability and reduced levels of junctional proteins at the membrane cell-cell interface along with reduced Cx-43 mRNA expression levels. Furthermore, this combination of stimuli determined an enhanced expression of the major markers of inflammation (TLR-4, IL-1β, IL-6, TNF-α, and IL-8) and others inflammatory mediators (P2X7R, VEGF-A, and ICAM-1) as well as the over-production of ROS. The pre-treatment with DHTS antagonized HG/BzATP stimulus and preserved the BRB integrity. In conclusion, the in vitro iBRB model demonstrated to be an useful tool for studying both the contribution of hyperglycemic conditions and P2X7R activation on iBRB dysfunction and for testing the therapeutic potential of DHTS in preventing and/or counteracting such alterations typical of BRB-related disorders, such as DR.