Abstract
Purpose :
Retinal vascular inflammation is a hallmark of early diabetic retinopathy (DR). Retinal endothelial cell (EC) activation contributes actively to vascular inflammation. We have previously identified lysyl oxidase (LOX)-dependent subendothelial matrix stiffening as a new determinant of retinal EC activation associated with early DR. But how LOX increases in diabetes remains unknown. Since advanced glycation end products (AGEs) are implicated in retinal EC activation and subendothelial matrix remodeling associated with DR, we asked whether their effects are mediated by LOX-dependent subendothelial matrix stiffening.
Methods :
Human retinal endothelial cells (HRECs) were treated with methylglyoxal (MGO, 10µM), a highly reactive dicarbonyl intermediate that promotes AGE formation, or high glucose (HG, 30mM) for 10 days. LOX activity was determined from the cell culture supernatant using Amplex® Red-based fluorescence assay while LOX protein and gene expressions were quantified by Western blotting & real time PCR, respectively. EC activation and subendothelial matrix markers including ICAM1, RAGE, collagen IV and fibronectin levels were assessed at protein and gene levels. The role of LOX in AGE-mediated effects was assessed by adding LOX inhibitor BAPN in MGO- and HG-treated HRECs.
Results :
Our findings reveal that MGO- or HG-treated HRECs exhibited a significant increase in LOX activity (p<0.05) and expression (p<0.05), which was accompanied by endothelial activation (increased ICAM1) & RAGE upregulation (p<0.05). Further, an increase in the levels of matrix proteins collagen IV & fibronectin was also observed. Importantly, LOX inhibition suppressed the effects of MGO or HG on HREC activation (p<0.05). Ongoing studies aim to use atomic force microscopy to quantify MGO-dependent matrix stiffness as well as distinguish between the direct effects of AGEs vs LOX-mediated effects of AGEs on matrix stiffness & HREC activation.
Conclusions :
These findings reveal a previously unknown and potentially crucial role of LOX in AGE-mediated retinal endothelial activation associated with DR. Importantly, these findings advocate the need to investigate the role of LOX-mediated vascular stiffening in the inflammatory effects of other DR risk factors and, thus, implicate LOX as a potentially new anti-inflammatory target for DR.
This is a 2020 ARVO Annual Meeting abstract.