Macrophage-derived WNTs in normal cardiac growth and regeneration following injury
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Date
01/12/2017Author
Castellan, Raphaël Fabrice Paul
Metadata
Abstract
Unlike other regenerative organs such as the liver, the adult mammalian heart does not
regenerate tissue lost following injury such as myocardial infarction (MI). Instead a
non-contractile fibrous scar develops that in the longer term leads to the development
of heart failure (HF). In contrast to the adult, neonatal mammals, including mice and
man, retain potent cardiac regenerative capacities and can replace myocardium lost
following injury. Understanding the mechanisms underlying scar free repair in the
neonate may help in development of new approaches to reduce the impact of
myocardial injury in adults. In this thesis MI was induced by coronary artery ligation
in mice at post-natal day 1 (P1). Novel electrocardiogram gated high resolution cardiac
ultrasound was developed to permit non-invasive confirmation of injury 1 day later
and regeneration 21 days later by loss, then restoration, of contractile function.
Macrophages (MФ) play important roles in organ growth and homeostasis, and are
required for scar-free regeneration of the neonatal mouse heart following MI. WNTs
are secreted lipophilic proteins with multiple roles in development. MФ-derived
WNTs are essential for scar free tissue regeneration following injury in the kidney,
liver, and gut, but their role in the heart is unknown. The primary aim of this thesis
was to investigate the role of MФ, and in particular MФ-derived WNTs in determining
normal growth of the myocardium from neonate to adult and also in regeneration of
the neonatal heart following injury.
In wild-type neonatal mouse hearts, Csf1r-expressing cells density (mostly
macrophages) was consistent across all time points studied. Three populations of
resident cardiac mononuclear phagocytes were identified by flow cytometry: F4/80hi,
CD11blo, Ly6C-ve - F4/80lo, CD11bhi, Ly6C-ve - F4/80lo, CD11bhi, Ly6C+ve. F4/80hi,
CD11blo, Ly6C-ve cells were hypothesised to correspond to yolk-sac derived
mononuclear phagocytes and F4/80lo, CD11bhi, Ly6C-ve - F4/80lo, CD11bhi, Ly6C+ve
to foetal liver/bone marrow derived mononuclear phagocytes. Three phases of
myocardial growth were identified by ultrasound and histological techniques:
hyperplastic (P2-P8, with increased Ki67 and cardiac troponin immunopositive cells),
hypertrophic/reorganisation (P8-P21, with increasing cardiomyocyte size and no
change in left ventricle wall thickness), and finally hypertrophic solely (P21-P42, with
increasing cardiomyocyte size and left ventricle wall thickness). Average coronary
vessel size was shown to decrease between P2 and P8 whilst vessel density was
increased. The number of α-smooth muscle actin (αSMA) coated vessels greatly
increased between P8 and P42, indicating vessel maturation. Throughout all phases
cardiac systolic function was maintained at steady state. Diastolic function was
however shown to mature from a foetal to an adult pattern between P2 and P8, with
reversal of the E:A wave ratio on Doppler ultrasound.
In mice globally deficient in MФ due to a germline knock-out of the Csf1r gene (Csf1rnull
mice), both body and heart weights were decreased from P7 onwards. The number
of proliferating (Ki67+ve) cardiomyocytes at P1 and P7 was unchanged in Csf1r-null
mice but there was a trend towards decreased cardiomyocyte size at P7, suggesting an
influence on hypertrophic rather than hyperplastic growth of the myocardium. There
was also a trend for slowed vascular network maturation, with a delay in the shift from
large to smaller vessels in hearts from Csf1r-null mice. In mice with MФ-directed
(Csf1r-icre mediated) depletion of Porcupine (Porcn), a gene encoding an enzyme
required for WNT acylation and secretion cardiac growth, vascularisation, fibrosis and
function were all similar in Cre-ve and Cre+ve animals until P41, when cardiomyocyte
size and cardiac systolic function were both significantly increased in Cre+ve animals.
However, the underlying mechanism is unknown.
In the neonatal mice, Csf1r expressing cells, mostly MФ, were identified in association
with regenerating myocardium after induction of MI at P1. Flow cytometry data
showed that by P7 the putative resident yolk-sac derived population had mostly
disappeared from the heart and was replaced by F4/80lo cells, similar to the pattern
reported in the adult. In the regenerating myocardium, Axin2 expression was increased
consistent with activation of canonical Wnt signalling. Expression of Wnt5b and Fzd2
receptor, both associated with fibrosis, was significantly increased relative to age
matched uninjured hearts. MФ-directed depletion of Porcn did not influence either the
functional decrease at day 1 or recovery at day 21 following induction of MI at P1.
Coronary re-vascularisation was also unaffected by the genotype. However, retention
of intra-myocardial fibrosis (picrosirius red staining) was significantly increased in
hearts at day 21 post-MI from mice with MФ-directed depletion of Porcn. MФ-derived
WNTs are therefore required for scar-free wound healing in the heart, as they are in
the liver and the kidney where they regulate matrix metalloproteinase activity.
In summary, novel ECG-gated high-resolution in vivo ultrasound developed in this
project has allowed characterisation of cardiac structure and function during early
post-natal growth and following injury and regeneration in neonatal mice. The resident
MФ population of the heart is established pre-natally, and may play a role in
determining maturation of the developing vascular network, although this does not
involve MФ-derived Wnt signalling. Following MI, the MФ population may expand
from bone marrow cells and MФ accumulate around the regenerating tissue. MФ
derived WNTs are not required for regeneration of the neonatal myocardium but do
have a role in ensuring scar free wound healing and this merits further investigation.