Phenotypic characterization of rabbit model for aortic valve stenosis: a novel medial calcification paradigm

Abstract Introduction Calcific aortic valve stenosis (CAVS) is the result of subtle, chronic inflammation and osteoblastic differentiation. As we lack human specimens of the early stages, reliable and reproducible animal models are needed to facilitate research. We previously demonstrated the ability of a novel rabbit CAVS vitamin D2 toxicity protocol to produce calcification and valve stenosis (1). We sought to characterize the phenotype of the model at the final stage. Methods Twelve New Zealand Rabbits were randomized 1:1 to control (normal chaw) and experimental group (normal chaw+1% cholesterol+3.500 I.U.s Vitamin D2, in oil in a biscuit) for 7 weeks. Animals were sacrificed and aortic valve cusps were snap frozen or formalin-fixed paraffin embedded. Cusps were then mechanically homogenized in buffer optimized for protein extraction and total protein measured with Bradford method. Part of the extract was subjected to trypsinization, in-gel digestion and untargeted LC-MS/MS. The rest was used to quantitate BMP-2 with total protein-normalized sandwitch competitive ELISA. Thin tissue sections were stained with Masson's trichrome, Von Kossa and H&E. Osteopontin, Bone sialoprotein II (BSPII), tissue non-specific alkaline phosphatase (TNAP) and osteocalcin (OCN) were detected on tissue with immunohistochemistry. Femoral bones from the same animals served as positive controls. Results Aortic valve cusp demonstrate large areas of collagen degradation and calcification in the medial layer, almost sparing the intima. Osteopontin deposits were colocalized with the calcification area in the media, whereas BSPII, TNAP and OCN were not expressed in the lesion, although present in bones. Similarly, BMP-2 levels were not significantly different between groups (experimental = 43.45 vs controls = 62.75 pg/ml, Mann-Whitney U test p=0.496). Proteomic analysis revealed a set of 96 differentially expressed proteins between cases and controls, interestingly including sortilin, osteonectin, beta-crystallin A2, Matrix Gla protein, Na/H exchanger 3, V-type H ATPase subunit D, Y-box binding protein. Conclusion The novel rabbit vitamin D2 toxicity protocol leads to excessive medial calcification of the aortic valve, with overexpression of osteopontin but without other classic markers of CAVS. Proteomics analysis reveals novel pathways with pathophysiological implications for the model and medial calcification. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Hellenic Cardiology Society, Hellenic Heart Foundation

Download Full-text

Modified New Zealand rabbit model produces severe aortic valve calcification and stenosis via extracellular membranous particles

European Heart Journal ◽

10.1093/ehjci/ehaa946.3722 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

N Anousakis-Vlachochristou ◽

A Varela ◽

M Kyriakidou ◽

S Parimalam ◽

S Badilescu ◽

...

Keyword(s):

New Zealand ◽

Aortic Valve ◽

Optical Imaging ◽

Aortic Valve Stenosis ◽

Rabbit Model ◽

Alcoholic Solution ◽

Funding Source ◽

Vitamin D2 ◽

Calcium Cations ◽

Ft Ir

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<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

Download Full-text

Investigation of the optimal rabbit model for aortic valve stenosis

European Heart Journal ◽

10.1093/ehjci/ehaa946.2318 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

N Anousakis-Vlachochristou ◽

M Katsa ◽

A Panara ◽

A Varela ◽

M Kyriakidou ◽

...

Keyword(s):

Aortic Valve ◽

Aortic Valve Stenosis ◽

Rabbit Model ◽

Public Institution ◽

Funding Source ◽

Vitamin D2 ◽

Group A ◽

Ft Ir ◽

Group B ◽

Group D

Abstract Background/Purpose Anatomically, hemodynamically relevant and validated animal models for aortic valve stenosis are of great need. Drolet rabbit model with tricuspid anatomy produced conflicting results for unclear reasons. We hypothesized that limitations concentrate in the regimen administration. We sought to evaluate multiple doses, ways of administration and time periods. Methods We included New Zealand rabbits in 4 groups: Group A (Drolet): was fed with normal chaw (nc)+0.5% cholesterol (chol)+3500 IUs Vitamin D2/kg (VD2, ergocalciferol, Sigma) in water daily for 12 weeks (wks), Group B: nc+0.5%chol+3500 IUs/kg VD2 in oil incorporated in a bisquit daily for 8 wks, Group C: nc+0.5%chol+8750 IUs/kg VD2 in oil-biscuit for 8 wks, Group D: nc+0.5%chol+17500 IUs VD2 in oil-biscuit for 8 wks vs controls (fed only with nc). After 12 and 8 wks the rabbits were sacrificed. Aortic valve area (AVA) and mean gradient (meanGr) were assessed with echocardiography (Vivid 7, M3S transducer, GE) and serum obtained, at baseline and before sacrifice. VD2 levels were evaluated through Chemiluminescent Microparticle Immuno Assay (CMIA, Abbott) and liquid chromatography – tandem mass spectrometry (LC-APCI-MS/MS). Animals received i.v. 18F-NaF one hour before sacrifice and valve was ex-vivo imaged with microPET/CT (Mediso nanoScan). Aortic cusps were analyzed with Fourier-Transformed Infrared Spectroscopy (FT-IR, Nicolet 6700 spectrometer, OMNIC 7.3 software). Valves from surgical patients with severe stenosis served for comparison purposes. Results In Group A at 12 wks AVA and meanGr remained unchanged but biomineralization was detected with FT-IR with vibrations in the region of 1800–800 cm–1 demonstrating the deposition of CaCO3 and non-hydroxyapatite Ca3(PO4)2 identical to human lesion. Calcification was detected on cusps with 18F-NaF. VD2 levels were out of upper detection range with CMIA due to cross reaction, whereas all samples measured through LC-MS/MS were below the detection limit of the method (<19,1 ng/mL). Significant Assessment heterogeneity (RSD=27%) was observed on VD2 water regimen. In Group B, AVA changed from 0.5 cm2 to 0.4 cm2 and meanGr increased from 1.1 to 2.1 mmHg, p<0.05 and in Group C AVA: 0.5 cm2 to 0.3 cm2 and meanGr: 1 to 2.95 mmHg, p<0.05, while VD2 serum concentration were 511 ng/mL. In Group D animals die unexpectedly at 2 weeks, with autopsy revealing massive myocardial hypertrophy of the left ventricle (LVH) without compromise of the aortic valve. Conclusions The modified diet produces aortic valve stenosis and biomineralization detectable with 18F-NaF, chemically identical to human lesion. Very high doses of Vitamin D2 directly produce LVH, possibly leading to arrythmiogenesis. The modified high-fat-vitamin D2 rabbit model proved suitable for translational research of aortic valve stenosis disease. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): National and Kapodistrian University of Athens

Download Full-text

Ramipril retards development of aortic valve stenosis in a rabbit model: mechanistic considerations

British Journal of Pharmacology ◽

10.1111/j.1476-5381.2010.01084.x ◽

2011 ◽

Vol 162 (3) ◽

pp. 722-732 ◽

Cited By ~ 27

Author(s):

Doan TM Ngo ◽

Irene Stafford ◽

Aaron L Sverdlov ◽

Weier Qi ◽

Ronald D Wuttke ◽

...

Keyword(s):

Aortic Valve ◽

Aortic Valve Stenosis ◽

Rabbit Model

Download Full-text

Beneficial Effects of High-Density Lipoproteins on Acquired von Willebrand Syndrome in Aortic Valve Stenosis

Thrombosis and Haemostasis ◽

10.1160/th17-10-0729 ◽

2018 ◽

Vol 118 (02) ◽

pp. 288-297 ◽

Cited By ~ 2

Author(s):

C. Gebhard ◽

F. Maafi ◽

B. Stähli ◽

A. Bonnefoy ◽

C. Gebhard ◽

...

Keyword(s):

Aortic Valve ◽

Aortic Valve Stenosis ◽

Rabbit Model ◽

Hdl Cholesterol ◽

Experimental Models ◽

High Density ◽

High Density Lipoproteins ◽

Major Protein ◽

Acquired Von Willebrand Syndrome ◽

Von Willebrand

Background Infusions of apolipoprotein A-I (apoA-I), the major protein component of high-density lipoproteins (HDL), result in aortic valve stenosis (AVS) regression in experimental models. Severe AVS can be complicated by acquired von Willebrand syndrome, a haemorrhagic disorder associated with loss of high-molecular-weight von Willebrand factor (vWF) multimers (HMWM), the latter being a consequence of increased shear stress and enhanced vWF-cleaving protease (ADAMTS-13) activity. Although antithrombotic actions of HDL have been described, its effects on ADAMTS-13 and vWF in AVS are unknown. Methods and Results We assessed ADAMTS-13 activity in plasma derived from a rabbit model of AVS (n = 29) as well as in plasma collected from 64 patients with severe AVS (age 65.0 ± 10.4 years, 44 males) undergoing aortic valve replacement (AVR). In both human and rabbit AVS plasma, ADAMTS-13 activity was higher than that in controls (p < 0.05). Accordingly, AVS patients had less HMWM than controls (66.3 ± 27.2% vs. 97.2 ± 24.1%, p < 0.0001). Both ADAMTS-13 activity and HMWM correlated significantly with aortic transvalvular gradients, thereby showing opposing correlations (r = 0.3, p = 0.018 and r = −0.4, p = 0.003, respectively). Administration of an apoA-I mimetic peptide reduced ADAMTS-13 activity in AVS rabbits as compared with the placebo group (2.0 ± 0.5 RFU/sec vs. 3.8 ± 0.4 RFU/sec, p < 0.05). Similarly, a negative correlation was found between ADAMTS-13 activity and HDL cholesterol levels in patients with AVS (r = −0.3, p = 0.045). Conclusion Our data indicate that HDL levels are associated with reduced ADAMTS-13 activity and increased HMWM. HDL-based therapies may reduce the haematologic abnormalities of the acquired von Willebrand syndrome in AVS.

Download Full-text