Abstract
Purpose :
The trabecular meshwork (TM) of the conventional outflow pathway participates in the regulation of outflow resistance, and TM dysfunction is the primary cause of elevated intraocular pressure (IOP) in glaucoma. Specifically, abnormal extracellular matrix (ECM) homeostasis by the TM contributes to outflow dysfunction, leading to a fibrotic/stiffer TM. In contrast, a healthy TM responds to elevated IOPs by remodeling ECM to facilitate increased outflow. We hypothesized that nano-sized extracellular vesicles (EVs) called “exosomes” are key participants in ECM homeostasis.
Methods :
Experiments were designed to determine the effect of mechanical stretch and glaucoma on the exosome proteome released from primary human TM cells. TM cells were isolated from glaucomatous (GTM, n=1) and non-glaucomatous (NTM, n=1) donors that were grown to confluence under normal cell culture conditions and then switched to EV-depleted media. NTM cells (n=3) were also subjected to mechanical stretch (20%) for 24 hours. Conditioned cell culture media was collected every 48-72 hours for up to 3 months, or immediately after cell stretch and purified exosomes were isolated using iodixanol buoyant density gradient centrifugation. Fractions (fraction numbers 5-10) were assessed using mass spectrometry, Western blot and nanoparticle tracking analysis (NTA).
Results :
Proteomic analysis of exosomes from TM cells showed an abundance of ECM-related proteins, including fibronectin, collagen and the integrin ligand EDIL3, which mediates cell binding and internalization of ECM proteins. Out of the top 20 proteins found across the cell strains, 72% were ECM-related. We validated fibronectin expression by Western blot, and found a 55% decrease in fibronectin binding to exosomes from GTM cells compared to NTM cells. NTM cells subjected to mechanical stretch released exosomes that bind 63% more fibronectin than unstretched controls. NTA demonstrated that the isolated exosomes from GTM cells appear to be larger than those from NTM cells; fractions 5-8: 103±11 vs 123.1±17nm and fractions 9-10: 112±1 vs 149±20nm.
Conclusions :
ECM proteins are major components of exosomes released from TM cells. Abundance of these proteins increase with mechanical stretching, suggesting a role in ECM opsonization, degradation and homeostasis. Importantly, our data also suggests that this process may be disrupted in GTM cells due to tissue stiffening or decreased exosome opsonization/binding.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.