Abstract
The epicardium is a mesothelial layer
surrounding the heart that is highly active during development and integral to
the formation of functional cardiac muscle. Epicardium-derived cells (EPDC)
populate the underlying myocardium with essential cells types via epithelial-to-mesenchymal
transition (EMT); a process whereby epithelial
cells transition into a more migratory and developmentally plastic mesenchymal state. Although
the epicardium is quiescent in adult heart, reactivation occurs following
hypoxic injury, characterised by EPDC expansion and trophic paracrine
signalling that contributes to neovascularisation. However, endogenous epicardium
reactivation it is not sufficient to compensate for damage to adult mammalian heart
and the development of therapeutic agents is required to augment this process. Characterisation
of EPDC signalling factors may therefore identify therapeutic targets that can
be amplified or attenuated via exogenous means for the treatment of cardiac
injury. Extracellular vesicles (EV) are nanoparticles secreted by cells that
transport molecular cargo that can modulate the phenotype of recipient cells. EVs
derived from numerous cardiac cell types have shown cardioprotective and
restorative effects when injected
in vivo
following myocardial infarct (MI). The impact of EPDC-derived EVs on the
adult heart has not been investigated. The aim of this study is therefore, firstly,
to assess the therapeutic efficacy of EPDC-derived EVs for treating MI; and
secondly, to develop a pipeline for identifying small compounds that attenuate
EV biogenesis and release.
Epithelial and mesenchymal EVs were isolated from
conditioned media of immortalised mouse EPDC (mEPDC) via differential centrifugation.
Samples were validated by Nanoparticle Tracking Analysis (NTA; Nanosight),
western blot and electron microscopy. EVs from mEPDC in
epithelial and mesenchymal states were enriched for the tetraspanins CD9 and
CD81, relative to whole source lysates. The functional impact of
epithelial-mEPDC-derived EVs (Epi-EPDC-EVs) and mesenchymal-mEPDC-derived EVs
(Mes-EPDC-EVs) on mEPDC was assessed via an automated wound heal assay, using
doses of 0.5 x 109, 1.0 x 109 and 3.0 x 109 EV
particles per well of a 96-well plate. Both
EV cohorts were found to impact wound closure capacity of mEPDCs. Epi-EPDC-EV treatment
had a dose dependent effect on rate of wound healing. Interestingly,
Mes-EPDC-EVs improved wound closure capacity at 0.5 x 109 and 1.0 x
109 particles per well but hindered healing at 3.0 x 109
particles per well.
These results demonstrate
that mEPDC secrete EVs in both the epithelial and mesenchymal states,
furthermore, EVs from both of these contexts elicit a functional impact on
mEPDCs. Interestingly, Epi-EPDC-EVs and Mes-EPDC-EVs elicited different effects
on recipient cells at the same dose. Further investigation is required into EV
cargo, effects on other cells types and
in
vivo efficacy. The development of a screening pipeline to assess the effect
of small compounds on EV dynamics may prove to be particular valuable for
translating EV-based therapies into the clinic.
Research
funded by The British Heart
Foundation Oxbridge Regenerative Medicine Centre (grant
reference: RM/17/2/33380) and
Oxstem Cardio.
