Large animal models of cardiovascular disease

Abstract

The human cardiovascular system is a complex arrangement of specialized structures with distinct functions. The molecular landscape, including the genome, transcriptome and proteome, is pivotal to the biological complexity of both normal and abnormal mammalian processes. Despite our advancing knowledge and understanding of cardiovascular disease (CVD) through the principal use of rodent models, this continues to be an increasing issue in today's world. For instance, as the ageing population increases, so does the incidence of heart valve dysfunction. This may be because of changes in molecular composition and structure of the extracellular matrix, or from the pathological process of vascular calcification in which bone-formation related factors cause ectopic mineralization. However, significant differences between mice and men exist in terms of cardiovascular anatomy, physiology and pathology. In contrast, large animal models can show considerably greater similarity to humans. Furthermore, precise and efficient genome editing techniques enable the generation of tailored models for translational research. These novel systems provide a huge potential for large animal models to investigate the regulatory factors and molecular pathways that contribute to CVD in vivo. In turn, this will help bridge the gap between basic science and clinical applications by facilitating the refinement of therapies for cardiovascular disease.

Keywords: Marfan syndrome; aortic stenosis; calcific aortic valve disease; cardiovascular disease; genetic engineering; large animal models; vascular calcification.

Figure 1

Simplified cross section of the aortic valve showing progression of aortic valve calcification. Valve endothelial cells (VECs) line the valve leaflet surface. The inner layers of the valve consist of the fibrosa, spongiosa and ventricularis. The principal cell type within each layer is the valve interstitial cells (VICs). The fibrosa contains collagen (Types I and III), the spongiosa contains glycosaminoglycans (GAGs) and the ventricularis—elastin fibres. In calcific aortic valve disease (CAVD), often the fibrosa layer becomes calcified and thickened. This may be because of lipid deposition and inflammatory processes which trigger the osteochondrogenic transdifferentiation of VICs. Calcium deposition then occurs, forming bone‐like material as neovascularization around the calcified lesions and remodelling of the extracellular matrix occurs. This results in the formation of calcified nodules and the thickening of the valve leaflet

Figure 2

Simplified diagram showing potential RANK/RANKL/OPG involvement in bone remodelling and in vascular calcification. Receptor activator of nuclear factor kappa‐B ligand (RANKL) from osteoblasts or endothelial cells binds to the Receptor Activator of Nuclear Factor kappa‐B (RANK) of osteoclast precursors, or vascular smooth muscle cells (VSMCs). This leads to differentiation into mature osteoclasts in the bone, which are involved in bone resorption, whereas in vascular calcification, VSMCs undergo a phenotypic transition into osteochondrogenic cells that can deposit mineralized matrix. Osteoprotegerin (OPG) is the decoy receptor for RANKL, and a potential inhibitor for mineralization

Figure 3

Simplified diagram showing potential NOTCH1 involvement in vascular calcification. NOTCH1 signalling may be involved in the inhibition, as well as the promotion, of vascular calcification through additional factors. Black arrows indicate stimulatory effects, whilst red lines indicate inhibitory effects

Figure 4

TALEN or CRISPR/Cas9 systems of gene editing. After zygote injection of customized transcription activator‐like effector nuclease (TALEN) mRNA or Cas9 mRNA/single guide RNA (sgRNA), the gene of interest can be targeted and cut to produce a double strand break (DSB). TALENs work in pairs as the FokI endonuclease requires dimerization in order to produce a DSB. Various CRISPR/Cas9 systems exist for different applications. Fundamentally, the sgRNA directs the Cas9 nuclease to the site of targeting. After a DSB is created, DNA repair mechanisms occur via two main pathways: non‐homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ leads to insertion/deletion mutations (indels), whilst HDR can be used to insert desired sequences. Through these mechanisms, an edited animal can be produced