Abstract
Purpose :
Trabecular meshwork (TM) biomechanics is important in regulating aqueous humor (AH) drainage and intraocular pressure (IOP). This study aims at understanding the role of activation and inhibition of the transcription factor SREBP which is the master regulator of lipid biogenesis on the regulation of TM extracellular matrix (ECM) content via the modulation of lipid synthesis.
Methods :
Quantitative-polymerase chain reaction (qPCR), immunofluorescence, immunoblot were performed on human TM (HTM) cells and porcine TM (PTM) cells in culture to assess - 1) expression of transcript variants of sterol regulatory-element binding protein (SREBP) and SREBP cleavage- activating protein (SCAP), and 2) effects of SREBP activation via clozapine, inhibition of SREBP-SCAP complex via fatostatin on - SREBP activation, regulation of ECM like fibronectin (FN), collagen (Col1A) in TM, SREBP target genes expression related to lipid biogenesis. Porcine anterior segment perfusion cultures were used to assess the effects of SREBP activation and inhibition on IOP changes. Student’s t-test and two-way ANOVA were used for statistical analyses and results were significant if p<0.05 with a sample size of n=3-5.
Results :
Transcript variants of SREBP-F1V1, V2, V3, and -F2V1 and SCAP were expressed in HTM and PTM cells. Activation of SREBP by clozapine significantly increased IOP (p=0.0440, n=5) after 17h clozapine perfusion. Clozapine treatment on HTM cells increased FN and Col1A fibril formation. Perfusion with fatostatin lowered IOP. HTM cells treated with fatostatin decreased ECM distribution and reduced gene expression of SREBP1, SREBP2, and their target genes involved in fatty acids, triglycerides, and cholesterol biosynthesis. Interestingly, we found a significant decrease in acetyl-CoA carboxylase (ACC) and low density lipoprotein receptor (LDLR) genes (p=0.0001, n=3).
Conclusions :
Our study identifies that modulating SREBP alters TM lipids and IOP. Of significance is the decrease in ACC which catalyzes the carboxylation of acetyl-CoA into malonyl-CoA, a rate-limiting step in fatty acid biosynthesis. Acetyl-CoA metabolism is implicated in primary open-angle glaucoma and ACC1 regulates ECM turnover and tissue fibrosis. Future studies on SREBP in TM will identify the potential regulation of membrane properties, AH outflow resistance, and novel therapeutics.
This is a 2021 ARVO Annual Meeting abstract.