Non-Pigmented Ciliary Epithelium Derived Exosomes and Their Role within the Drainage System as a Pharmacological Intervention Target for Glaucoma

Student thesis: Doctoral Thesis

Abstract

Background: Glaucoma is a progressive optic neuropathy characterized by a specific pattern of
optic nerve head and visual field impairment. Damage to the visual system in glaucoma is due to
the death of the retinal ganglion cells, the axons of which comprise the optic nerve and carry the
visual impulses from the eye to the brain. The number of people aged 40–80 years with glaucoma
is expected to increase to 76.0 million by 2020 and 111.8 million by 2040. Primary open-angle
glaucoma (POAG) is the most common form of the disease and there is currently no
cure. Raised intraocular pressure (IOP) is the major risk factor in the development of POAG and
the reduction of IOP is known to limit disease onset and slow disease progression. The balance
between Aqueous humour (AH) production by the Non-Pigmented Ciliary Epithelium (NPCE) and
drainage through the Trabecular Meshwork (TM) maintains IOP, thus any increase in the
production or impaired drainage of AH can lead to a rise in the IOP. It has previously been
proposed that a cross-talk communication may exist between inflow tissue and outflow tissues
through potential modulators in AH. These modulators include neuroproteins, microRNAs,
hormones and extracellular vesicles (EVs). Among EVs, exosomes are of particular interest
because of their role in cell-to-cell communication. Exosomes are 30-150 nm nanoparticles that
are released from most cell types under normal and pathological conditions. Nucleic acids and
proteins embedded in the exosome membrane are composed of phospholipids. Rich exosomal
cargos have potential to regulate gene expression, cell death, oxidative stress, cellular
metabolism and signal transduction pathways. Once released, exosomes can be internalized by
neighboring or distal cells and equip cells with the ability to influence cellular function. Limited
data, however, is available regarding the role of exosomes on Wnt signaling in the outflow system
in ocular physiology and pathology conditions. Numerous studies over the past several years
indicate that the rich cargo content in the exosomes has the potential to regulate various cellular
processes including gene transcription, oxidative stress, cell death, differentiation and signal
transduction. Among the many signaling pathways affected by exosomes, the Wnt signaling has
emerged as a critical regulator of AH outflow facility. Wnt signaling and its effector β-catenin
regulate the expression of genes encoding extracellular matrix (ECM) proteins. Increase in the
collagen content of the ECM may lead to TM obstruction and decrease of AH outflow. In addition, β-catenin binds with cadherin proteins to bridge between the cytoplasmic domain of cadherin
and the cytoskeleton. Thus, modulation of Wnt signaling by exosomes may provide a useful tool
in the reorganization of cytoskeleton, ECM of TM cells, and regulation of IOP. Oxidative stress is
identified to be an important risk factor in the development of glaucoma. Several studies have
demonstrated the ability of exosomes to regulate the oxidative stress via modulating ROS
production. ROS promotes stabilization of β-catenin, suggesting a link between oxidative stress
and canonical Wnt signaling. The role of exosomes released under oxidative stress conditions in
TM and their effects on Wnt signaling remain unknown.
PhD Thesis Hypothesis: We assume that there are communication processes taking place
between the NPCE cells and TM cells via NPCE exosomes affecting Wnt and PI3K/AKT signaling in
TM cells and causing alterations in the stress response of TM cells.
The General Goals of This Research are:
Characterization of exosomes excreted from NPCE cell lines as well as exosomes derived from
primary NPCE cells.
Investigation of the effects of the NPCE exosomes on TM cells Wnt pathway-associated genes and their protein expression.
To investigate the biological effect of exosomes released from NPCE cells under oxidative stress
condition on TM molecular responses to oxidative stress.
Results: In this study, we demonstrated that NPCE cells release EVs that carry classic exosome
markers including Tsg 101 and Alix on their surface. EVs comprised a membrane bilayer and had
a round shape with size range characteristic of exosomes (50-140nm). In the exosome samples
we identified 584 miRNAs and 182 proteins involved in cell adhesion, cell signaling, cytoskeleton
regulation and oxidative stress. Using isothermal titration calorimetry technique, NPCE exosomes
were shown to interact with TM cells. NPCE exosomes internalized and transferred their cargo,
including microRNAs and proteins, to the TM recipient cells. Moreover, TM exosomes had the
capacity to selectively accumulate NPCE exosomes as compared to other control cell lines. To
test whether exosomes derived from NPCE cells influence the Wnt signaling in TM cells, the exosomes were co-cultured with TM cells. After 2 hour of exosome incubation we found
significant decreases in the expression of the Wnt signaling proteins and genes in TM cells
including β-catenin, p-GSKβ, AXIN2 and LEF1. We showed that NPCE exosome treatment
downregulated the expression of positive GSKβ regulator – AKT protein but increased the levels
of GSKβ negative regulator - PP2A protein in TM cells. In addition, we observed a significant
decrease in the levels of the Collagen3A and Cadherins in TM cells following NPCE exosomes
exposure, which have been shown to be important in the IOP homeostasis. Finally, the ability of
exosomes to deliver oxidative stress (OS) signal messages between NPCE and the TM cells was
studied. TM cells exposed to OS resulted in a significant 25% reduction in viability. When treated
with exosomes isolated from oxidative stressed NPCE CELLS (Ox-EVs) the viability decrease was
abolished. Increase in Nrf2 cytosolic and nucleic expression was found following Ox-EVs
treatment. We revealed that Ox-EVs regulate antioxidant gene expression, as exposure of TM
cells to Ox-EVs led to a significant increase in SOD2, GPx, HOX1 and NRF2 gene expression, while
this effect was not observed following normal exosome treatment (N-EVs). In addition, we
demonstrate that OX-EVs treatment resulted in significantly higher antioxidant capacity vs N-EVs
or untreated TM cells. The two major antioxidant enzymes, superoxide dismutase and catalase
activity were significantly higher following Ox-EVs treatment. Conclusions: Our findings suggest a pivotal role for exosomes derived from NPCE cells in
modulating canonical Wnt signaling in TM cells. These findings may have therapeutic relevance
since canonical Wnt pathway is involved in IOP regulation. Further investigation of NPCE-TM
communication by NPCE exosomes demonstrates the ability of exosomes to modulate OS
response in TM cells resulting in a better antioxidant capacity. This phenomenon can be the result
of exosome cargo modification under OS including proteins and miRNAs or/and oxidized
proteins, lipid and nucleic acids carried by the exosomes as cargo or on their surface. We suggest
that controlling antioxidant enzymes activity confers a therapeutic advantage for TM cells in
glaucoma. Further understanding of NPCE-derived exosome-responsive signaling pathways may
reveal new targets for pharmacological intervention within the drainage system as a target for
glaucoma therapy.
Date of Award2019
Original languageEnglish
SupervisorElie Beit - Yannai (Supervisor) & Sofia Sara Schreiber-Avissar (Supervisor)

Keywords

  • POAG
  • oxidative stress
  • TM cells
  • NPCE cells
  • Exosomes
  • Extracellular vesicles
  • Wnt signaling

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