Modified New Zealand rabbit model produces severe aortic valve calcification and stenosis via extracellular membranous particles
Authors: Anousakis-Vlachochristou N , Varela A , Kyriakidou M , Parimalam S , Badilescu S , Agapaki A , Lali D , Kapelouzou A , Anastassopoulou I , Packirisamy M , Anagnostopoulos C , Cokkinos D , Tousoulis D , Toutouzas K 
Published: November 2020

Abstract

Background/Purpose
In aortic valve stenosis calcification begins with nucleation on extracellular vesicles. In order to study early-stage disease, validated animal models are needed. The Drolet rabbit model is relevant due to tricuspid valve, but failed to consistently produce stenosis probably due to regimen administration. We compared a modified rabbit model and investigated the mechanisms and patterns of calcification.

Methods
New Zealand rabbits introduced to normal chaw+1% cholesterol+8750 IUs Vitamin D2/kg (Sigma) daily, in olive oil given in a bisquit vs control animals, for 8 weeks. Aortic valve area (AVA) and mean gradient (meanGr) was assessed with echocardiography (Vivid 7, M3S transducer, GE). At 8 weeks animals were sacrificed and valves were snap-frozen to −80°C. From each animal, one cusp was analyzed with Fourier-Transformed Infrared Spectroscopy (FT-IR, Nicolet 6700 spectrometer, OMNIC 7.3 software), another cusp was processed in alcoholic solution and the third was fixed 0.5 μm thin on 4% PFA; supernatant and tissue respectively examined with multispectral optical imaging. Valves from patients with severe stenosis were used for qualitative comparisons.

Results
At 8 weeks versus baseline, AVA reduced (0.5 cm2 to 0.3 cm2) and meanGr increased (1.1 to 2.95 mmHg, p&lt;0.05), in control was unchanged. FT-IR vibrations in the region of 1800–800 cm–1 demonstrated changes in the protein structure and deposition of CaCO3 and non-hydroxyapatite Ca3(PO4)2 identical to patients' lesions. Multispectral optical imaging of supernatants revealed numerous membranous particles and conductivity analysis indicated calcium cations accumulation on the phospholipids of membrane. The tissue images confirmed the degradations and dendrimer-like depositions of calcium cations most likely on carbonates of amino acids.

Conclusions
The modified high-fat-vitamin D2 rabbit model produces aortic valve stenosis, with chemically identical mineralization to human lesion. Multispectral photonics demonstrate the presence of calcified membranous extracellular particles, a hallmark of cardiovascular calcification. Dendrimer-like depositions correspond to growing deposits. The model is suitable as a research platform purposed for aortic valve stenosis.
Figure 1. A: Image from alcoholic solution supernatant. The bright spots have high conductivity due to Ca 2+ deposition. B: ImageJ surface plot of circulated region confirms calcification. C: 3D-plot illustrates mineralization of membranes. D: 3D-plot of human aortic valve. E: Hypermicroscopic image of rabbit valve tissue: dendrimer-like and mineral cation deposits.

Funding Acknowledgement
Type of funding source: Public Institution(s). Main funding source(s): National and Kapodistrian University of Athens, Greece; Concordia University, Montreal, Canada