Abstract
Purpose :
Endothelial cell (EC) activation is a major determinant of retinal vascular inflammation, a hallmark of early diabetic retinopathy (DR). Recent work from our lab has introduced a new paradigm for retinal EC activation in DR that identifies lysyl oxidase (LOX)-dependent retinal ‘vascular stiffening’ as a crucial regulator of retinal endothelial NF-kB activation, ICAM-1 upregulation, and leukocyte-EC adhesion in diabetes. Given this ability of LOX to mechanically regulate retinal EC activation via an increase in vascular stiffness, here we asked whether LOX can simultaneously exert a direct biochemical effect on retinal ECs, independently and/or by mediating the proinflammatory effects of key diabetic stressors viz. VEGF and TNFα.
Methods :
Human retinal endothelial cells (HRECs) were treated with recombinant VEGF (25 ng/mL) or TNF-α (10 ng/mL) for 4h and recombinant LOX (1 µg/mL) for 16h. mRNA Levels of LOX, ICAM-1, VEGFR-2 and TNFR-1 were quantified by RT-qPCR. Finally, the role of LOX in VEGF- and TNF-α-induced proinflammatory effects was assessed by addition of β-aminopropionitrile (BAPN, an inhibitor of LOX activity) 2h prior to VEGF or TNF-α stimulation.
Results :
Our findings revealed that VEGF- and TNF-α-treated HRECs exhibit a concomitant increase in ICAM-1 (by 1.4-fold; p<0.01) and LOX (by 1.3-fold; p<0.01) mRNA. Importantly, LOX inhibition (using BAPN) blocked the VEGF- and TNF-α -induced ICAM-1 upregulation in HRECs, thus indicating that LOX mediates the proinflammatory effect of VEGF and TNFα. Further, LOX inhibition prevented the VEGF- and TNF-α-induced upregulation of their corresponding receptors viz., VEGFR-2 and TNFR-1. That LOX actively promotes proinflammatory receptor expression was confirmed when recombinant LOX increased VEGFR-2 (by 1.7-fold; p<0.001), TNFR-1 (by 1.7-fold; p<0.001), and ICAM-1 (by 1.7-fold; p<0.01) expression in HRECs.
Conclusions :
These findings reveal a previously unknown but important proinflammatory role of LOX wherein it mediates the proinflammatory effects of VEGF and TNFα on HRECs by enhancing their receptor expression. Ongoing studies aim to determine the role of LOX in VEGF-/TNF-induced leukocyte-EC adhesion in vitro and simultaneously validate the aforementioned findings in a mouse model of DR. Successful completion of these studies has important translation implications as it may implicate LOX as a new anti-inflammatory target for effective DR management.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.