Abstract
Purpose :
Leukocyte adhesion to ICAM-1-expressing retinal vascular endothelial cells (EC) is a critical early step in diabetic retinopathy (DR). The adherent (activated) leukocytes release various factors to further amplify inflammation. Since we have recently shown that high glucose-induced vascular stiffening promotes leukocyte-EC adhesion, here we tested the hypothesis that adherent leukocytes also contribute to progressive stiffening of retinal vessels in diabetes, thereby amplifying retinal vascular inflammation associated with DR.
Methods :
Human U937 monocytes were activated with high glucose and PMA (200nM). Monocyte activation was confirmed by staining with DCF, a reactive oxygen species (ROS) indicator. Conditioned medium (CM) from activated monocytes was then added to human retinal ECs (HRECs), followed by qPCR analysis of ICAM-1, lysyl oxidase (LOX), and ROCK2 mRNA, key pro-inflammatory and matrix/EC stiffening mediators, respectively. We next used Amplex Red reagent to determine whether activated monocytes themselves undergo LOX upregulation. Finally, these biochemical measurements were correlated with atomic force microscopy (AFM) measurement and immunofluorescent staining of HREC stiffness and collagen IV matrix deposition, respectively.
Results :
High glucose and PMA treatment caused significant activation of U937 monocytes, as judged by a >10-fold increase in ROS levels. Importantly, HRECs treated with monocyte CM exhibited a marked increase in ICAM-1, LOX and ROCK2 mRNA expression. Our AFM measurements confirmed that activated monocytes cause a significant increase in EC stiffness, which correlates with increased endothelial LOX and ROCK2 mRNA. Interestingly, the activated monocytes themselves exhibited a two-fold increase in LOX levels. Finally, our preliminary studies indicate that activated monocytes cause an increase in collagen IV matrix deposition by HRECs. Studies are underway to better understand the molecular mechanisms by which activated monocytes contribute to LOX-dependent retinal vascular stiffening and inflammation.
Conclusions :
Our findings indicate a potentially crucial role of activated monocytes in vascular stiffening-dependent retinal inflammation associated with DR and provide rationale to further examine this pathway for the identification of novel anti-inflammatory targets for improved DR therapies in the future.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.