Alex Herman, Lauren Carville, Jasenka Fuchs-Guduric, Varun Pathak, Tim Curtis, Eleni Beli
Background: Endothelial cells (ECs) in the retinal vasculature act as essential gatekeepers, restricting inflammatory cell entry into the retina. In diabetes, circulating monocytes and granulocytes are elevated, driving chronic low‑grade inflammation. This inflammatory milieu increases adhesion molecule expression on retinal ECs, promoting leukostasis and leukocyte infiltration into the retinal parenchyma—key contributors to the development and progression of diabetic retinopathy. Previous work has shown that ECs derived from diabetic patients display disrupted circadian rhythmicity and elevated ICAM‑1 and VEGF expression, while patient fibroblasts exhibit altered temporal ICAM‑1 expression. Our group has also demonstrated that leukocyte recruitment into the retina follows a time‑of‑day rhythm, suggesting that circadian regulation of adhesion molecules may be central to controlling inflammatory cell entry in diabetes.
Methods: Primary human retinal endothelial cells (HRECs) were exposed to stimuli mimicking the diabetic microenvironment: high glucose (25 mM D‑glucose), inflammation (0.1 ng/ml TNF‑α), oxidative stress (50 μM H₂O₂), or a combination of these factors. Adhesion molecule expression (ICAM‑1, VCAM‑1, P‑selectin) was quantified using RT‑PCR. To assess the role of the circadian clock, Bmal1 was silenced in HRECs, and changes in adhesion molecule expression were measured by RT‑PCR and flow cytometry. Circadian rhythmicity was monitored using Per2‑luciferase bioluminescence recordings to determine how diabetic‑like stimuli alter endothelial clock function. Experiments were performed twice with n=3 technical replicates. Statistical analysis was done with GraphPad (2 Way ANOVA or cosinor for determination of circadian rhythmicity, p<0.05)
Results: High glucose or oxidative stress alone did not significantly increase ICAM‑1 or VCAM‑1 expression, whereas TNF‑α robustly induced ICAM‑1. Notably, combined treatment, reflecting the multifactorial nature of the diabetic microenvironment, produced a greater increase in adhesion molecule expression than any single stimulus. Silencing BMAL1 resulted in a marked reduction of ICAM‑1 at both mRNA and protein levels, indicating that ICAM‑1 expression is directly influenced by the endothelial circadian clock. Per2‑luciferase recordings revealed that combination treatment disrupted circadian rhythmicity, inducing a phase shift in HREC molecular clock oscillations.
Conclusion: These findings demonstrate that the diabetic microenvironment synergistically enhances adhesion molecule expression in retinal endothelial cells and disrupts intrinsic circadian rhythms. The reduction of ICAM‑1 following BMAL1 deletion highlights a direct regulatory role of the endothelial clock in controlling leukocyte adhesion pathways. Together, this work suggests that circadian dysregulation contributes to excessive leukocyte recruitment in diabetic retinopathy and may represent a novel therapeutic target for anti‑inflammatory intervention.