Tonabersat Inhibits Connexin43 Hemichannel Opening and Inflammasome Activation in an In Vitro Retinal Epithelial Cell Model of Diabetic Retinopathy

This study was undertaken to evaluate the connexin hemichannel blocker tonabersat for the inhibition of inflammasome activation and use as a potential treatment for diabetic retinopathy. Human retinal pigment epithelial cells (ARPE-19) were stimulated with hyperglycemia and the inflammatory cytokines IL-1β and TNFα in order to mimic diabetic retinopathy molecular signs in vitro. Immunohistochemistry was used to evaluate the effect of tonabersat treatment on NLRP3, NLRP1, and cleaved caspase-1 expression and distribution. A Luminex cytokine release assay was performed to determine whether tonabersat affected proinflammatory cytokine release. NLRP1 was not activated in ARPE-19 cells, and IL-18 was not produced under disease conditions. However, NLRP3 and cleaved caspase-1 complex formation increased with hyperglycemia and cytokine challenge but was inhibited by tonabersat treatment. It also prevented the release of proinflammatory cytokines IL-1β, VEGF, and IL-6. Tonabersat therefore has the potential to reduce inflammasome-mediated inflammation in diabetic retinopathy

Systemic Inflammasome Biomarkers as Predictors of Diabetic Retinopathy Progression: Evidence from a Pilot Study

The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) protein 3 (NLRP3) inflammasome pathway is believed to mediate chronic inflammation in diabetic retinopathy (DR); however, its impact on the progression of DR remains to be elucidated. Therefore, the primary aim of this pilot study was to determine whether systemic inflammasome biomarkers interleukin (IL)-1β and IL-18 can be used to predict DR progression. DR screening results were analyzed against weight, level of glycated hemoglobin (HbA1c), and plasma levels of inflammasome biomarkers (IL-1β and IL-18), as well as general inflammation markers (C-reactive protein (CRP), IL-6, IL-8, tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF)) in patients with type 2 diabetes at baseline and 1 year post-bariatric surgery. Cross-sectional analysis demonstrated that weight, HbA1c, CRP, and IL-18 did not correlate with DR severity. The progressed group showed a higher relative change in IL-18 and CRP levels compared to the stable and regressed groups. Furthermore, relative changes in plasma CRP levels correlated with those of IL-18. Although further validation with larger cohorts is necessary, this pilot study supports the hypothesis that systemic inflammasome activation is associated with DR progression

Orally Delivered Connexin43 Hemichannel Blocker, Tonabersat, Inhibits Vascular Breakdown and Inflammasome Activation in a Mouse Model of Diabetic Retinopathy

Diabetic retinopathy (DR), a microvascular complication of diabetes, is associated with pronounced inflammation arising from the activation of a nucleotide-binding and oligomerization domain-like receptor (NLR) protein 3 (NLRP3) inflammasome. Cell culture models have shown that a connexin43 hemichannel blocker can prevent inflammasome activation in DR. The aim of this study was to evaluate the ocular safety and efficacy of tonabersat, an orally bioavailable connexin43 hemichannel blocker, to protect against DR signs in an inflammatory non-obese diabetic (NOD) DR mouse model. For retina safety studies, tonabersat was applied to retinal pigment epithelial (ARPE-19) cells or given orally to control NOD mice in the absence of any other stimuli. For efficacy studies, either tonabersat or a vehicle was given orally to the inflammatory NOD mouse model two hours before an intravitreal injection of pro-inflammatory cytokines, interleukin-1 beta, and tumour necrosis factor-alpha. Fundus and optical coherence tomography images were acquired at the baseline as well as at 2- and 7-day timepoints to assess microvascular abnormalities and sub-retinal fluid accumulation. Retinal inflammation and inflammasome activation were also assessed using immunohistochemistry. Tonabersat did not have any effect on ARPE-19 cells or control NOD mouse retinas in the absence of other stimuli. However, the tonabersat treatment in the inflammatory NOD mice significantly reduced macrovascular abnormalities, hyperreflective foci, sub-retinal fluid accumulation, vascular leak, inflammation, and inflammasome activation. These findings suggest that tonabersat may be a safe and effective treatment for DR

Characterization of NLRP3 Inflammasome Activation in the Onset of Diabetic Retinopathy

The aim of this study was to characterize the role of nucleotide-binding oligomerization domain- (NOD-) like receptor (NLR) protein 3 (NLRP3) inflammasome activation in the onset of diabetic retinopathy (DR) using retina and vitreous from donors without diabetes mellitus (CTL), with diabetes mellitus alone (DM), and with DR. Retinal expression of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1), the key markers of retinal inflammation, connexin43 (Cx43) which is involved in upstream inflammasome regulation, as well as NLRP3 and cleaved caspase-1, the main markers of inflammasome activation, were evaluated using immunohistochemistry and Western blotting. Vitreous interleukin (IL)-1β and IL-18, biomarkers of the activated inflammasome, were measured using a Luminex multiplex assay. Results showed a significant increase in the number and size of Iba-1+ cells and NLRP3 expression in DM, while a significant increase in GFAP, Cx43, cleaved caspase-1 and vitreous IL-18, as well as a further increase in Iba-1 and NLRP3 was found in DR. This suggests that the inflammasome is already primed in DM before its activation in DR. Furthermore, IL-18 may act as the major effector of inflammasome activation in DR while nuclear translocation of cleaved caspase-1 may play a role in gene transcription contributing to DR onset

Immunohistochemical Characterization of Connexin43 Expression in a Mouse Model of Diabetic Retinopathy and in Human Donor Retinas

Diabetic retinopathy (DR) develops due to hyperglycemia and inflammation-induced vascular disruptions in the retina with connexin43 expression patterns in the disease still debated. Here, the effects of hyperglycemia and inflammation on connexin43 expression in vitro in a mouse model of DR and in human donor tissues were evaluated. Primary human retinal microvascular endothelial cells (hRMECs) were exposed to high glucose (HG; 25 mM) or pro-inflammatory cytokines IL-1β and TNF-α (10 ng/mL each) or both before assessing connexin43 expression. Additionally, connexin43, glial fibrillary acidic protein (GFAP), and plasmalemma vesicular associated protein (PLVAP) were labeled in wild-type (C57BL/6), Akita (diabetic), and Akimba (DR) mouse retinas. Finally, connexin43 and GFAP expression in donor retinas with confirmed DR was compared to age-matched controls. Co-application of HG and cytokines increased connexin43 expression in hRMECs in line with results seen in mice, with no significant difference in connexin43 or GFAP expression in Akita but higher expression in Akimba compared to wild-type mice. On PLVAP-positive vessels, connexin43 was higher in Akimba but unchanged in Akita compared to wild-type mice. Connexin43 expression appeared higher in donor retinas with confirmed DR compared to age-matched controls, similar to the distribution seen in Akimba mice and correlating with the in vitro results. Although connexin43 expression seems reduced in diabetes, hyperglycemia and inflammation present in the pathology of DR seem to increase connexin43 expression, suggesting a causal role of connexin43 channels in the disease progression

Intravitreal pro-inflammatory cytokines in non-obese diabetic mice: Modelling signs of diabetic retinopathy.

Diabetic retinopathy is a vascular disease of the retina characterised by hyperglycaemic and inflammatory processes. Most animal models of diabetic retinopathy are hyperglycaemia-only models that do not account for the significant role that inflammation plays in the development of the disease. In the present study, we present data on the establishment of a new animal model of diabetic retinopathy that incorporates both hyperglycaemia and inflammation. We hypothesized that inflammation may trigger and worsen the development of diabetic retinopathy in a hyperglycaemic environment. Pro-inflammatory cytokines, IL-1β and TNF-α, were therefore injected into the vitreous of non-obese diabetic (NOD) mice. CD1 mice were used as same genetic background controls. Fundus and optical coherence tomography images were obtained before (day 0) as well as on days 2 and 7 after intravitreal cytokine injection to assess vessel dilation and beading, retinal and vitreous hyper-reflective foci and retinal thickness. Astrogliosis and microgliosis were assessed using immunohistochemistry. Results showed that intravitreal cytokines induced vessel dilation, beading, severe vitreous hyper-reflective foci, retinal oedema, increased astrogliosis and microglia upregulation in diabetic NOD mice. Intravitreal injection of inflammatory cytokines into the eyes of diabetic mice therefore appears to provide a new model of diabetic retinopathy that could be used for the study of disease progression and treatment strategies