Role of ANLN in post ischaemic angiogenesis

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.