Abstract
Purpose :
Pseudoexfoliation glaucoma (PEXG), the ocular manifestation of pseudoexfoliation syndrome, is the most common identifiable form of open angle-glaucoma worldwide. Genetic risk variants for PEXG are found in lysyl oxidase-like 1 (LOXL1), an enzyme that crosslinks collagen/elastin and is critical for extracellular matrix (ECM) formation/repair. In addition, TGFβ1, a pro-fibrotic factor, is elevated in the aqueous humor of PEXG patients. The aim of this study is to investigate the interplay between LOXL1 and TGFβ1 in conventional outflow biology.
Methods :
Intravitreal injections of adenovirus encoding TGFβ1223-225 Ad5-CMV-TGFβ1(Ad-TGFβ1), or Ad5-CMV-GFP (Ad-GFP) as control were used to induce the expression of the active form of TGFβ1 in tissues of the conventional outflow pathway in C57BL/6 wild type mice (2 and 6-month-old) and 2-month-old C57BL/6xBalbC (CB6F1) Loxl1-/- (and Loxl1+/+). IOP was measured twice a week and outflow facility determined by iPerfusion. In parallel studies in primary human TM cells, LOXL1 was knocked down using small interference RNA, and then cells were treated with TGFβ1 (5ng/mL). TGFβ1 signaling and ECM remodeling/deposition modulators were determined by qPCR and western blotting.
Results :
For 2-month-old C57BL/6 mice, IOP increased (ΔIOP=5.36±3.01 mmHg, n=11) 7 days after injection with Ad-TGFβ1 and outflow facilities were reduced compared to Ad-GFP (1.85±0.77 vs 2.33±1.20 nl/min/mmHg). For 6-month-old C57BL/6 mice, IOP increased (ΔIOP=5.29±1.60 mmHg, n=7) 12 days after treatment and facilities were reduced (2.16±0.64 vs 2.59±0.63 nl/min/mmHg). In the CB6F1 Loxl1+/+ mice, Ad-TGFβ1 caused IOP to dramatically increase (ΔIOP=10.00±7.68 mmHg, n=5), while Loxl1-/- mice appeared resistant (ΔIOP= -0.67 ±4.16 mmHg, n=3); although variability in responses of control mice to TGFβ1 and limited numbers of healthy Loxl1-/- mice prevented statistical significance (p=0.24). In vitro, TGFβ1 stimulated the mRNA expression of LOXL1 (p=0.04), TGFβRI (p<0.0001), BMP1 (p=0.006), and protein expression of FN (p=0.006) and α-SMA (p=0.03) in TM cells. Significantly, knockdown of LOXL1 in TM cells inhibited all of these effects (p<0.01), aside from BMP1 upregulation.
Conclusions :
These results suggest that LOXL1 expression levels in conventional outflow tissues determine IOP responses to active TGFβ1, play a role in the regulation of fibrosis and have implications for ocular hypertension in PEXG.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.