Abstract
Purpose :
Trabecular meshwork (TM) tissue is subjected to constant mechanical stretch due to ocular pulse created by the cardiac cycle. This triggers the modification of various intracellular signaling responses to counter mechanical insults. A loss of such response can lead to elevated intraocular pressure (IOP), a major risk factor for primary open-angle glaucoma. The purpose of this study was to understand the metabolic response of TM cells to mechanical stretch.
Methods :
Normal primary human TM cells (n=3) in culture were subjected to 15% mechanical stretch, 1 cycle/second, for 8h and 24h using a computer-controlled Flexcell unit, and unstretched cells were controls. We profiled for mRNA changes in 8h samples using qPCR and protein changes using mass spectrometry-based quantitative proteomics on 24h samples. The data was statistically significant if the p≤ 0.05 for mRNA using paired Student’s t-test and proteins were screened out using criteria, p ≤ 0.05 and mean ± 2σ of log2 as confidence fold change limits.
Results :
Cyclic mechanical stretch significantly increased 9 proteins and decreased 31 proteins including those involved in metabolism. Among those, were the proteins involved in cholesterol biogenesis, which showed significant changes (Fig 1)- levels of squalene synthase, a critical enzyme in the cholesterol biosynthetic pathway significantly increased (p=0.04) with an increasing trend in HMG CoA synthase (HMGCS), and HMG CoA reductase (HMGCR) and a significant decrease in Kelch-like ECH-associated protein 1 (KEAP1) (p=0.04), which is a negative regulator of nuclear factor-E2-related factor 2 (Nrf2) involved in sterol metabolism. Further examining the mRNA transcripts of genes involved in cholesterol biosynthesis machinery, there was a significant upregulation of sterol regulatory element-binding protein 2 (SREBP2) (p=0.05), a transcription factor and the master regulator of sterol pathway, as well as HMGCS (p=0.03), and HMGCR (p=0.03) genes.
Conclusions :
This is a first-hand report on comparative analysis between changes in mRNA and proteins in response to mechanical stretch. We find that TM cells respond to the changes in biomechanical properties by orchestrating the transcriptional control of cholesterol biosynthesis and tightly regulating the enzymes in the sterol synthesis pathway.
This is a 2021 ARVO Annual Meeting abstract.