Identifying Novel Therapies to Prevent Atherosclerosis

Aims

To further investigate a role for the CLEC14A/endosialin/multimerin complex in atherosclerosis and potential therapeutic intervention.

Background

We recently showed CLEC14A to be expressed in regions of low shear stress (Mura et al., 2012) and particularly in athero-prone regions of the large arteries (Figure, Bicknell unpublished data). Two earlier studies implicate CLEC14A in having a role in atherosclerosis. The Stockholm study of atherosclerotic tissue from 40 patients identified six key genes of which one was CLEC14A (Hagg et al., 2009). A second study looked for genes contributing to carotid atherosclerosis in mice and of the nine identified one was again CLEC14A (Grainger et al., 2017). These studies prompted us to examine plaque formation in the ApoE/CLEC14A double knock out mouse. The figure shows a marked reduction in plaque formation compared to control ApoE mice.

An independent study recently showed that the closely related C-type lectin endosialin/CD248 also promotes atherosclerosis (Hasanov et al. 2017). We have identified the ligand for CLEC14A as the endothelial specific extracellular protein multimerin-2 (Khan et al., 2017). We have further shown that MMRN2 is the long sought after ligand for endosialin (Khan et al, 2017) although they bind to MMRN2 in different sites. Thus, we have identified a ligand receptor complex spanning the fibroblast/endothelial junction where knockout of each of the two ligands phenocopies effects on atherosclerosis.

Hypothesis

That the MMRN2 complex with CLEC14A and endosialin is a major promoter of atherosclerosis and its disruption will open new therapeutic approaches.

Experimental Methods and Research Plan

Experimental Methods:
DNA cloning, protein expression, mouse models of disease, genetic alteration of mice using CRISPR, mouse pathology, histochemical analysis

Further validation of the pro-atherogenic role of CLEC14A

We will examine atherosclerotic lesions in total aortas for increased contractile VSMC markers in the absence of CLEC14A by immunohistochemistry and gene expression array (Agilent) to confirm that the phenocopy seen in the KO mice is due to them signalling through a single complex.

We will examine atherosclerosis in a second in house complementary animal model involving implantation of a shear stress modifying cast around the carotid artery to generate atherosclerotic lesions. Lesions will be examined for expression of contractile and leukocyte (e.g. CD68, macrophage) markers by immunohistochemistry, FACS analysis and gene expression arrays (Agilent).

Direct Evidence of a role for MMRN2:

We will edit out the two binding regions of multimerin-2 separately by CRISPR in mice.

Does disruption of the MMRN2 complex block atherosclerosis:

1. Antibodies. We will first test in simple models of acute inflammation such as peritonitis, for example, leukocyte recruitment (neutrophils and monocytes) following challenge with zymosan. Our C4 antibody to CLEC14A blocks MMRN2 binding to CLEC14A (Noy et al. 2015). C4 blocks angiogenesis and tumour growth in vivo. Preliminary evidence suggests that the C4 antibody blocks plaque formation (Bicknell, Rainger et al. unpublished). Rat anti-mouse endosialin antibodies are available and will be screened.

2. Peptide antagonists. The peptide binding region from MMRN2 that binds to CLEC14A blocks angiogenesis in vivo and will be studied alongside the complementary region for endosialin for athero blocking activity

3. Vaccination. The aim will be to vaccinate (see Zhuang and Bicknell, 2016) against CLEC14A to prevent atherosclerosis.

Expected outcomes and impact

There are at least two opportunities for high impact publications arising from this work. If as it looks, targeting CLEC14A and or endosialin does indeed reduce the plaque burden this could lead to a high impact publication in journal such as J. Clin. Invest. or J. Exp. Med.