Role of ANLN in post ischaemic angiogenesis
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Date
25/11/2019Item status
Restricted AccessEmbargo end date
25/11/2020Author
Cathcart, Benjamin James
Metadata
Abstract
Angiogenesis is a key physiological process by which new blood vessels are dynamically
formed in Endothelial cell (EC) driven processes. The identification of new potential genes of
interest in ECs can help to further understand the underlying mechanisms of angiogenesis
and help develop new therapeutic strategies for cardiovascular disease. One such gene
identified in the Caporali lab is Anillin (ANLN) an F-actin binding protein with functions in
cytokinesis, asymmetric cell division and potential roles in migration and proliferation in
multiple cell types.
We identified that ANLN was highly upregulated in the ECs of a mouse model of hind limb
ischaemia. In vitro modulation of ANLN expression with siRNA and ANLN-EGFP lentivirus in
HUVECs identified an effect of ANLN expression on multiple aspects of EC function including,
migration wound healing and barrier function. A correlation between ANLN expression and
tubulogenesis assay performance was observed associating ANLN with an early model of
sprouting angiogenesis. A potential regulatory mechanism of ANLN by ZFP36 RNA binding
proteins was identified in vascular inflammation and ischaemia in vivo and in vitro. Based on
in vitro observations, ANLN expression was investigated in the ECs of the developing
vasculature, and in a model of vascular injury using the anln:anln-eGFP x flk:mcherry reporter
zebrafish. ANLN was not associated with the 24 hpf developing vasculature in the trunk, CNS
or tail of the fish however there was a strong band of Anln+ve cells near the developing trunk
vasculature and caudal artery plexus. A micro point laser injury of the caudal artery was
developed; with injuries being recoverable 24 hours post injury and causing association of
neutrophils with injury sites. Anln was not observed in ECs of the caudal artery after laser
injury, however, increased Anln+ve cells are observed in mechanical injury of the tail fin
identifying Anln expression in a response to injury. Hypomorphic modulation of anln
expression by morpholino injection of flk:gfp embryos did not alter the vascular morphology
of the 2-3 dpf zebrafish vasculature.
This data for the first time associates ANLN with the EC response to the angiogenic response
in ischaemia. Although our zebrafish study was mostly negative a new method for inducing
vascular injury in zebrafish was developed and there was a novel application of an anln:anlneGFP
zebrafish for the investigation of ANLN in vascular regeneration. Our vascular injury
model for the investigation of ANLN can be improved by utilising a mechanical method of
vessel injury for further studies. Application of vascular specific CRISPR/CAS9 mutation of
ANLN expression may improve identification of a vascular phenotype in zebrafish, which was
not observed with morpholino injection. Further in vitro experiments should focus on ANLN-EGFP
in sprouting angiogenesis models and investigating a potential nuclear role of ANLN.