We aim to evaluate the AK in regulating adenosine

We aim to evaluate the AK in regulating adenosine signaling in the retina. It was reported that the degree of BAY 61-3606 synthesis injury directly depends on expression levels of AK and the resulting extracellular levels of adenosine (Boison, 2006). Indeed, transgenic mice overexpressing AK are highly susceptible to stroke-induced brain injury (Shen et al., 2011). We therefore hypothesized that adenosine kinase inhibitors (AKI) could play the same protective role in the diabetic retina.
Methods
Results
Discussion Biochemical studies have shown that inflammatory reactions (Joussen et al., 2004), including TNF-α release, are relatively early events that occur in response to diabetes before vascular dysfunction involving acellular capillary formation and neovascularization (Kern and Barber, 2008). Moreover, TNF-α has been shown to recruit leukocytes, cause vascular breakdown and promote neuronal injury at high levels (Joussen et al., 2009). Thus, treatments targeting early features of DR would provide long-term vascular benefits. Adenosine released at inflamed sites exhibits anti-inflammatory effects through A2AAR (Bong et al., 1996). Although adenosine and its agonists are protective in animal models of inflammation, their therapeutic application has been limited by systemic side effects such as hypotension, bradycardia, and sedation (Williams, 1996). Moreover, adenosine usually disappears very rapidly in physiological or inflammatory conditions due to rapid reuptake and subsequent intracellular metabolism (Möser et al., 1989). The use of AK inhibitors represents one possible way to amplify the endogenous therapeutic effects of site- and event-specific accumulation of extracellular adenosine while minimizing hemodynamic toxicity. Endogenous adenosine levels in the brain are mainly dependent on the activity of AK, the key enzyme of adenosine metabolism (Gouder et al., 2004). This notion is based on several lines of evidence: 1) transgenic mice overexpressing AK are highly susceptible to stroke-induced brain injury (Pignataro et al., 2007); 2) pharmacological inhibition of AK provides seizure suppression in various models of epilepsy (Ugarkar et al., 2000); 3) inhibition of AK in hippocampal slices increases endogenous adenosine and depresses neuronal firing, whereas inhibition of adenosine deaminase has little or no influence (Huber et al., 2001); 4) AK activity is regulated in response to brain injury and is subject to developmental regulation (Studer et al., 2006, Pignataro et al., 2008). We demonstrated that AK has the same importance in the retina. In the present work, intraperitoneal injection of ABT 702 was found to cause a significant inhibition of ICAM-1 and TNF-α mRNA as well as protein levels in the retina of diabetic mice, suggesting the curative effect of ABT 702 on inflammation associated with STZ-diabetic model. ABT 702 also prevented up-regulation of Iba1; supporting the hypothesis that ABT 702 reduces retinal inflammation through attenuation of microglia activation. Following this, we used primary culture of rat retinal microglial cells to gain insights into the mechanism of ABT 702's anti-inflammatory effect. The results indicate that treatment of ABT 702 inhibited AGA-induced TNF-α release. Furthermore, ABT 702 was more effective than ADA inhibitor in inhibiting TNF-α release, suggesting a major role for AK in the regulation of extracellular adenosine. The ability of ABT 702 to mitigate AGA-induced TNF-α release suggests the importance of inhibiting AK activity in ameliorating this inflammatory response through increasing adenosine levels. To test this hypothesis, the inhibitory effect of ABT 702 on AGA-induced TNF-α release was examined in the presence of AR subtype-selective antagonists in the retinal microglial cells. This inhibitory effect was successfully blocked only by 4- 2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-α][1,3,5]triazin-5-ylamino]ethyl phenol (ZM 241385), a selective A2AAR antagonist. These results suggest that ABT 702 inhibits AGA-induced TNF-α release in retinal microglia through A2AAR. A2AAR mediates the suppressive effects of adenosine in macrophages as well as microglial cells (Kreckler et al., 2006).