Abstract
Purpose :
Based on an unbiased and serendipitous finding, we recently showed that cyclic mechanical stretch of human trabecular meshwork (HTM) cells increased SREBP2 activation and modified lipid synthesis. This prompted us to further understand the role of transcriptional factor SREBPs, the master regulators of lipid biogenesis, on IOP regulation and TM biomechanics.
Methods :
To assess the effects of IOP changes upon SREBPs inactivation – a) Porcine anterior segment (PAS) was perfused with a specific SREBPs inactivator fatostatin for 24h ex vivo with IOP changes constantly monitored and b) SREBP cleavage-activating protein (SCAP)-floxed (SCAPf/f) mice were intravitreally injected with adenovirus carrying Cre (AdCre-eGFP) to conditionally knockdown SCAP and inhibit SREBPs activation in TM in vivo and IOP was measured before and after injection compared to Ad-eGFP-, saline-injected and untouched control groups. To assess the effects of SREBPs inactivation on HTM cultures in vitro using fatostatin, qPCR, immunofluorescence, and immunoblot were done for SREBPs, SREBPs responsive lipogenic genes, extracellular matrix (ECM) remodeling, actin polymerization, and focal adhesions (FAs) localization. Student’s t-test and two-way ANOVA were used for statistics and results were significant if p<0.05 with a sample size of 4-10.
Results :
Inactivation of SREBPs significantly lowered IOP in – a) PAS after 12h fatostatin perfusion (p=0.04) and sustained until 22h, b) the AdCre-eGFP group (12.97±0.71mmHg) compared to untouched (19.67±0.83mmHg, p=0.0001), saline (20.5±1.00mmHg, p=0.0001), and AdeGFP (21.74±1.47mmHg, p=0.0001) groups by 10 days after injection and maintained until 50 days. HTM cells treated with fatostatin significantly – 1) reduced the expression of active-SREBP1 (p=0.03) and -SREBP2 (p=0.0001) screened from nuclear fractions, and their responsive lipogenic genes (p<0.05), 2) attenuated ECM proteins like fibronectin (FN) (p=0.01) and collagen (COL1A) (p=0.001) production and secretion, 3) decreased F-actin fibers and actin regulators like fascin1 (p=0.03) and LIM domain kinase 1 (LIMK1) (p=0.04), and FAs distribution.
Conclusions :
Our study identifies that inactivating SREBPs significantly lowered IOP potentially by decreasing TM lipid content, ECM accumulation, and contractility. This provides an innovative therapeutic target for future glaucoma treatment.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.