
==== Front
Animal Model Exp Med
Animal Model Exp Med
10.1002/(ISSN)2576-2095
AME2
Animal Models and Experimental Medicine
2096-5451
2576-2095
John Wiley and Sons Inc. Hoboken

38372410
10.1002/ame2.12393
AME212393
AMEM-2023-0115.R3
Original Article
Regular Article
Original Article
A novel mouse model of calcific aortic valve stenosis
Qian et al.
Qian Ningjing https://orcid.org/0000-0002-8707-2757
1 2 3
Wang Yaping https://orcid.org/0000-0002-2740-1731
1 2 3
Hu Wangxing https://orcid.org/0000-0002-7065-4822
1 2 3
Cao Naifang https://orcid.org/0009-0000-9315-0822
1 2 3
Qian Yi https://orcid.org/0000-0001-8054-6887
2 3 4
Chen Jinyong https://orcid.org/0000-0003-3359-7811
1 2 3
Fang Juan https://orcid.org/0000-0002-0900-3844
5
Xu Dilin https://orcid.org/0009-0003-4507-3393
1 2 3
Hu Haochang https://orcid.org/0000-0003-3798-1773
1 2 3
Yang Shuangshuang https://orcid.org/0009-0007-2510-2117
1 2 3
Zhou Dao https://orcid.org/0000-0001-7815-9490
1 2 3
Dai Hanyi https://orcid.org/0000-0002-7782-891X
1 2 3
Wei Dongdong https://orcid.org/0000-0002-3082-8138
4
Wang Jian'an https://orcid.org/0000-0002-4583-3204
1 2 3 6 wangjianan111@zju.edu.cn

Liu Xianbao https://orcid.org/0000-0003-1556-9198
1 2 3 liuxb@zju.edu.cn

1 Department of Cardiology The Second Affiliated Hospital, Zhejiang University School of Medicine Hangzhou China
2 State Key Laboratory of Transvascular Implantation Devices China
3 Cardiovascular Key Laboratory of Zhejiang Province Hangzhou China
4 Department of Cardiovascular Surgery The Second Affiliated Hospital, Zhejiang University School of Medicine Hangzhou China
5 Department of Endocrinology The Second Affiliated Hospital, Zhejiang University School of Medicine Hangzhou China
6 Binjiang Institute of Zhejiang University Hangzhou China
* Correspondence
Xianbao Liu and Jian'an Wang, Department of Cardiology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Email: liuxb@zju.edu.cn and wangjianan111@zju.edu.cn

19 2 2024
8 2024
7 4 10.1002/ame2.v7.4 Themed Issue: Study on Cardiovascular and Cerebrovascular Diseases 523532
11 10 2023
16 1 2024
© 2024 The Authors. Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

Abstract

Background

Calcific aortic valve stenosis (CAVS) is one of the most challenging heart diseases in clinical with rapidly increasing prevalence. However, study of the mechanism and treatment of CAVS is hampered by the lack of suitable, robust and efficient models that develop hemodynamically significant stenosis and typical calcium deposition. Here, we aim to establish a mouse model to mimic the development and features of CAVS.

Methods

The model was established via aortic valve wire injury (AVWI) combined with vitamin D subcutaneous injected in wild type C57/BL6 mice. Serial transthoracic echocardiography was applied to evaluate aortic jet peak velocity and mean gradient. Histopathological specimens were collected and examined in respect of valve thickening, calcium deposition, collagen accumulation, osteogenic differentiation and inflammation.

Results

Serial transthoracic echocardiography revealed that aortic jet peak velocity and mean gradient increased from 7 days post model establishment in a time dependent manner and tended to be stable at 28 days. Compared with the sham group, simple AVWI or the vitamin D group, the hybrid model group showed typical pathological features of CAVS, including hemodynamic alterations, increased aortic valve thickening, calcium deposition, collagen accumulation at 28 days. In addition, osteogenic differentiation, fibrosis and inflammation, which play critical roles in the development of CAVS, were observed in the hybrid model.

Conclusions

We established a novel mouse model of CAVS that could be induced efficiently, robustly and economically, and without genetic intervention. It provides a fast track to explore the underlying mechanisms of CAVS and to identify more effective pharmacological targets.

Here we established a novel mouse model of calcific aortic valve stenosis that could be induced efficiently, robustly and economically, and without genetic intervention. Via induction by wire injury combined with vitamin D injection, the model allowed wild type mice to develop the typical features of calcific aortic valve stenosis, calcium deposition in valve leaflets and hemodynamically significant stenosis, as well as histopathological changes in multi‐dimensions.

animal model
calcific aortic valve stenosis
valve calcification
National Natural Science Foundation of China 10.13039/501100001809 81770252 82030014 82271606 U22A20267 Binjiang Institute of Zhejiang UniversityZY202205SMKY001 Key Program of Major Science and Technology Projects in Zhejiang Province2021C03097 2022C03063 source-schema-version-number2.0
cover-dateAugust 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:03.09.2024
Qian N , Wang Y , Hu W , et al. A novel mouse model of calcific aortic valve stenosis. Anim Models Exp Med. 2024;7 :523‐532. doi:10.1002/ame2.12393
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pmc1 INTRODUCTION

Calcific aortic valve stenosis (CAVS) is one of the most common valvular heart diseases. 1 , 2 CAVS is characterized by calcification and fibrosis of leaflets leading to valve thickening and stiffness and eventually progression to valve stenosis. As its prevalence has increased, CAVS has become a serious social and economic burden globally and an important population‐wide health problem. However, currently there are no medical therapies that can prevent disease progression. 3 , 4 The root of the problem is that the basic mechanisms of CAVS remain opaque. Therefore, constructing reliable and efficient models to uncover the underlying mechanisms ought to be a research priority.

Over the years, many mouse models have been developed to study CAVS, of which the most commonly used are hyperlipidemic and atherosclerotic mutant mouse models. 5 , 6 Some transgenic mouse models, for example, Notch1‐null mice, have also been developed to simulate the formation of CAVS. 7 , 8 , 9 However, all these models suffer the drawback that they do not adequately characterize the functional and histopathological changes unique to CAVS, specifically, typical calcium deposition in valve leaflets and hemodynamically significant stenosis. In addition, these models have some practical limitations, such as being costly and time consuming to establish, and difficult to manipulate genetically. 10

The aim of this study was to develop and characterize a mouse model of CAVS that directly mimics the typical features of CAVS, namely calcium deposition in valve leaflets and hemodynamically significant stenosis, as well as histopathological changes in multi‐dimensions such as fibrosis and inflammation, in a simple, robust, efficient, and universally applicable way.

2 METHODS

2.1 Animals and experimental design

Male wild type mice (C57BL/6 background) aged 6–8 weeks were obtained from Shanghai Laboratory Animal Research Center (Shanghai, China) and maintained at the Laboratory Animal Center, Seconded Affiliated Hospital of Zhejiang University. The mice were randomly assigned to four study groups: (1) sham‐operation group; (2) sham‐operation with vitamin D group; (3) aortic valve wire injury (AVWI) group; and (4) AVWI with vitamin D group (Figure 1A). All mice were fed with a normal diet. The animal experiment protocols were approved by the Institutional Animal Research Committee of Zhejiang University (2021‐NO.009) and performed according to institutional guidelines and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

FIGURE 1 Schematic representation of the experiments. (A) The wild type mice were randomly assigned to four study groups: (1) sham‐operation group; (92) sham‐operation with vitamin D group; (3) aortic valve wire injury (AVWI) group; and (4) AVWI with vitamin D group. Vitamin D was subcutaneously injected on 3 consecutive days from the second day after AVWI or sham‐operation. Transthoracic echocardiography was performed serially at 7, 14, 28 days post AVWI or sham‐operation. (B and C) For AVWI, the wire was firstly inserted into the left ventricle under echocardiographic guidance. Then the wire was pulled back and positioned at the left ventricular outlet, just below the aortic valve level.

2.2 Establishment of CAVS models

AVWI was performed as described previously. 11 , 12 In brief, mice were anesthetized with pentobarbital sodium (60 mg/kg) by intraperitoneal injection. A tip‐bent guide wire (ASAHI INTECC MIRACLEbros 3) was introduced into the right carotid artery after exposing it by blunt dissection. Then the wire was carefully passed over the aortic valve and advanced to the left ventricle under echocardiographic guidance (Figure 1B). The wire was pulled back and positioned at the left ventricular outlet, just below the aortic valve level (Figure 1C). Valve injury was induced by scratching heart tissue 20 times with the wire and then spinning the wire 100 times in situ. The wire was retracted and the right carotid artery was ligated after the procedure. The sham‐operation group underwent the same procedure without advancing the wire across the aortic valve. Vitamin D (500, 000 IU/kg body weight; Cat. C9756; Sigma‐Aldrich), dissolved in corn oil, was subcutaneously injected on 3 consecutive days under gas anesthesia with 1.5% isoflurane from the second day of AVWI or sham‐operation. 13 In the other two groups, the same volume of corn oil was injected as control.

2.3 Echocardiography

Transthoracic echocardiography was performed serially under 1.5% isoflurane anesthesia using a Vevo 3100 imaging system equipped with a 40‐MHz transducer (VisualSonics, Toronto, AB, Canada). Cardiac parameters and ventricular function were assessed in parasternal long axis and parasternal short axis views using B mode and M mode. Color Doppler and continuous wave Doppler modes were used to evaluate aortic valve function by quantification of the aortic valve outflow tract in suprasternal section views, which was adjusted to align with the parasternal long axis, with the transducer set at an appropriate angle. The cardiac package built in VisualSonic system was used to measure and calculate all echocardiographic parameters.

2.4 Histological analysis

The mice were sacrificed at the appointed time post AVWI or sham‐operation and the whole hearts were collected after perfusion with phosphate buffered saline (PBS). The whole hearts were embedded in optimum cutting temperature compound (OCT; BDH Laboratory Supplies, Safat, Kuwait) and later sectioned into 6 μm serial sections for histological staining and immunofluorescence. The sections of aortic valve were routinely stained with hematoxylin and eosin (HE) according to standard protocols to measure the area of aortic valve leaflets. For visualization of the fibrosis, Masson's trichrome staining was performed with a Masson's trichrome staining kit (Solarbio, Beijing, China) according to the manufacturer's instruction. For visualization of the calcification, the 4% paraformaldehyde fixed sections were air‐dried, washed free of OCT and stained by Alizarin Red staining (Oricell, Guangzhou, China) for 10 minutes, followed by washing and sealing. Images were obtained using a Leica fluorescence microscope (Leica, Germany). The images were quantified by ImageJ software (NIH, Bethesda, MD, USA).

2.5 Immunofluorescence

For immunofluorescence staining, cryosections were first fixed in 4% paraformaldehyde for 10 minutes. The cryosections were then incubated in 0.1% Triton X‐100 for 10 minutes and blocked with 3% bovine serum albumin (BSA, AMRESCO, Solon, OH, USA) for another 1 hour. The following primary antibodies were used for an overnight incubation: RUNX2 (1:200 dilution, ab192256, Abcam), BMP2 (1:200 dilution, ab214821, Abcam), CD68 (1:200 dilution, ab53444, Abcam), SP7 (1:200 dilution, ab209484, Abcam), α‐SMA (1:400 dilution, A5228, Sigma) and VCAM1 (1:50 dilution, ET1601‐18, Huabio). The slides were washed and incubated with the corresponding second antibody: goat anti‐rat IgG H&L (Alexa Fluor® 488) (1:400 dilution, ab150165, Abcam), goat anti‐mouse IgG H&L (Alexa Fluor® 488) (1:400 dilution, A‐11001, Invitrogen), donkey anti‐rabbit IgG H&L (DyLight 550) (1:400 dilution, ab96892, Abcam), and donkey anti‐mouse IgG H&L (DyLight 550) (1:400 dilution, ab96876, Abcam). DAPI (H‐1200‐10, Vector Laboratories, USA) was used to counterstain nuclei. Images were acquired using a Leica fluorescence microscope (Leica, Germany) and quantified by ImageJ software (NIH, Bethesda, MD, USA).

2.6 Statistical analysis

All statistical analysis was performed with SPSS version 24.0 and GraphPad Prism version 8.0.1. Continuous variables were presented as means ± standard deviation (SD) with checks for normal distribution and categorical variables were presented as counts with percentages. For continuous variables, differences between groups were determined by unpaired Student's t test for two groups and one‐way analysis of variance (ANOVA) for three or more groups followed by Tukey's test for multiple comparisons. p < 0.05 was set as the statistically significant level.

3 RESULTS

3.1 Establishment of the model and survival of the mice

To investigate and characterize the hybrid model, mice were randomized into four groups with 12 mice in each group: a sham group, a simple vitamin D group, a simple AVWI group, and a hybrid model group in which AVWI was combined with vitamin D. Mice in the simple AVWI group, and the hybrid model group underwent aortic valve injury induced by a wire inserted through the right carotid artery into the left ventricle. The mice in the sham group and the simple vitamin D group underwent the same procedure, but without advancing the wire across the aortic valve. A total of 8 mice died during operation or within the first 24 h after it, including 2 in the sham group, 2 in the simple vitamin D group, 3 in the simple AVWI group, and 1 in the hybrid model group. There was no statistically significant difference in terms of the mortality rate among the groups. The major cause of death was puncture point bleeding of the right carotid artery due to an unintentional operation error or the ligature falling off. The remaining 10 mice in the simple vitamin D group, and 11 mice in the hybrid model group were subcutaneously injected with vitamin D on 3 consecutive days. And the remaining 10 mice in the sham group, and 9 mice in the simple AVWI were injected with the same volume of corn oil. These mice all survived at 28 days.

3.2 The hybrid model induces CAVS with significant hemodynamic alterations

Color Doppler and continuous wave Doppler modes were performed at 7, 14, 28 days post model establishment to evaluate hemodynamic performance of the aortic valve. Establishment of the hybrid model significantly increased aortic jet peak velocity and mean gradient from 7 days (Figure 2A,B). The aortic jet peak velocity and mean gradient continued to increase in a time dependent manner and tended to be stable at 28 days (hybrid model group vs. sham group, aortic jet peak velocity, 4214.81 ± 555.45 vs. 1631.33 ± 551.23 mm/s, p < 0.0001; mean gradient, 20.81 ± 5.14 vs. 2.41 ± 1.39 mmHg, p < 0.0001; n = 10, 11) (Figure 2A–D). Additionally, aortic jet peak velocity and mean gradient were upregulated in simple AVWI model mice compared with the sham group at 28 days, but were lower than the AVWI and vitamin D hybrid model (Figure 2C,D). A mild increase in aortic jet peak velocity and mean gradient was seen in the simple vitamin D model compared to the sham group (Figure 2C,D).

FIGURE 2 Hemodynamic alterations and cardiac function post model establishment. Upper left panels, Aortic jet peak velocity (A) and mean gradient (B) of AVWI with vitamin D group mice at baseline, 7, 14, 28 days post model establishment (n = 11). Upper right panels, Aortic jet peak velocity (C) and mean gradient (D) of the four groups mice at 28 days post model establishment (n = 9–11). Middle and lower panels, Heart weight (HW)/body weight (BW), heart rate, ejection fraction, fractional shortening, left ventricular internal dimension in diastole (LVID, d) and left ventricular internal dimension in systole (LVID, s) of sham group, vitamin D group, AVWI group, and AVWI with vitamin D group mice (n = 9–11). *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

3.3 The hybrid model leads to aortic valve thickening, calcification and collagen accumulation

Morphological and histological evaluation were performed on aortic valves from the four groups. H&E staining indicated that the area of the aortic valve was significantly greater in the hybrid model group compared with that of the sham group at 28 days (Figure 3A,B). The simple AVWI group showed an increase in aortic valve area compared with the sham group, while the simple vitamin D group did not develop signs of valve thickening. Alizarin Red staining was applied to visualize the calcification of valve leaflets. Distinct calcification was detected in the aortic valve of the hybrid model group at 14 days post model establishment and became more significant at 28 days (Figure 3C,D). The simple vitamin D group also showed an increased positive area with Alizarin Red staining, but calcium deposition was more clearly observed in the hybrid model group at 28 days post model establishment (Figure 3E,F). Masson's trichrome staining further revealed large amounts of collagen accumulation in the aortic valve of the hybrid model group as well as the simple AVWI group (Figure 3G,H). There were no signs of notable collagen accumulation in the aortic valves of the simple vitamin D group.

FIGURE 3 Histological analyses of aortic valve post model establishment. (A) Representative H&E staining images of sham group, vitamin D group, aortic valve wire injury (AVWI) group and AVWI with vitamin D group mice at 28 days post model establishment. (B) Quantitative analysis of valve leaflet area (n = 6) at 28 days post model establishment. (C) Representative Alizarin Red staining images of AVWI with vitamin D group mice at baseline, 7, 14, 28 days post model establishment. (D) Quantitative analysis of calcium deposition of AVWI with vitamin D group mice at baseline, 7, 14, 28 days post model establishment (n = 6). (E) Representative Alizarin Red staining images of sham group, vitamin D group, AVWI group and AVWI with vitamin D group mice at 28 days post model establishment. (F) Quantitative analysis of calcium deposition (n = 6) at 28 days post model establishment. (G) Representative Masson's trichrome staining (Masson) images of sham group, vitamin D group, AVWI group and AVWI with vitamin D group mice at 28 days post model establishment. (H) Quantitative analysis of collagen volume fraction (n = 6) at 28 days post model establishment. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

3.4 The hybrid model initiates osteogenic differentiation of the aortic valve

To characterize the calcification process, immunofluorescence of osteogenic differentiation markers was performed in aortic valves collected at 28 days from the four groups. Consistent with the increased calcium deposition, the expression level of BMP2 was markedly upregulated in the aortic valve of the hybrid model group but was not significantly increased in the simple vitamin D group or AVWI group when compared with the sham group (Figure 4A,B). The expression level of RUNX2 was lowest in the sham group and showed a significantly increase in the hybrid model group (Figure 4C,D). The simple vitamin D group and AVWI group showed trends to increases in RUNX2 expression compared with the sham group but the changes did not reach significant levels. SP7, also known as Osterix, a bone specific transcription factor required for osteogenic differentiation, also stained positively in the histological calcified sections, especially in the hybrid model group (Figure 4E,F).

FIGURE 4 Osteogenic differentiation of aortic valve post model establishment. (A, C, and E) Representative immunofluorescence staining images of osteogenic markers, including BMP2, RUNX2 and SP7 in aortic valve of sham group, vitamin D group, aortic valve wire injury (AVWI) group and AVWI with vitamin D group mice at 28 days post model establishment. (B, D, and F) Quantitative analysis of BMP2, RUNX2 and SP7 expression (intensity) in aortic valve of sham group, vitamin D group, AVWI group and AVWI with vitamin D group mice at 28 days post model establishment (n = 6). *p < 0.05; **p < 0.01; ****p < 0.0001.

3.5 The hybrid model induces inflammation and fibrosis

Prolonged inflammation is also supposed to be one of the critical factors in the process of CAVS. Immunofluorescence revealed there was obvious upregulation of CD68 (Figure 5A,B) and VCAM1 (Figure 5C,D) expression levels in the aortic valve of the hybrid model group, which suggested activation of the inflammation response and macrophage infiltration of the valve leaflets. Moreover, since increased collagen accumulation was observed in the hybrid model group, we checked the expression of αSMA in the aortic valves. Consistent with the Masson's trichrome staining results, immunofluorescence showed a significantly increased level of αSMA (Figure 5E,F) in the hybrid model group, which confirmed the fibrotic activation process.

FIGURE 5 Inflammation and fibrosis of aortic valve post model establishment. (A, C, and E) Representative immunofluorescence staining images of CD68, VCAM1 and αSMA in aortic valve of sham group, vitamin D group, aortic valve wire injury (AVWI) group and AVWI with vitamin D group mice at 28 days post model establishment. (B, D, and F) Quantitative analysis of CD68, VCAM1 and αSMA expression (intensity) in aortic valve of sham group, vitamin D group, AVWI group and AVWI with vitamin D group mice at 28 days post model establishment (n = 6). *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

4 DISCUSSION

This study establishes a novel hybrid mouse model of CAVS in a robust and convenient way. Here, we show that vitamin D injection combined with AVWI in wild type mice leads to the development of CAVS and simulates the classical structural and hemodynamical changes seen with CAVS. The model mimics the clinical and pathological features of CAVS, including high mechanical stress, calcification, fibrosis, endothelial injury and inflammation of valve leaflets.

CAVS is one of the most challenging heart diseases and its characteristics of calcification, thickening and restricted leaflet mobility are usually detected by multi‐modal imaging. 1 The development of relevant animal models of CAVS is extremely important for further investigation of the pathophysiology and molecular mechanisms and for the evaluation of potential therapies. Despite the developments of the past few years, there is still an unmet demand for better animal models of CAVs to better understand the disease. Several potential animal models have been developed, including mouse, rat, rabbit and pig models, with the majority being mouse models. 14 Briefly, these can be summarized as diet‐induced models, wire injury models and genetic models. The most commonly used model, a high fat, high cholesterol diet‐induced model in hyperlipidemic and atherosclerotic mutant mice, is non‐invasive and convenient to establish. However, it is a time‐consuming model, with the animals frequently requiring over six months to develop significant valve thickening. Moreover, the characteristic aortic stenosis defined by hemodynamic changes and histologically revealed increased calcification are generally hard to trigger. The wire injury model has been recently developed and has become a promising model with the advantage that it models hemodynamic changes and valve thickening well. 11 , 12 Despite this, we found in our previous study 15 that in this model the calcification phenotype of the aortic valve was poorly characterized, which was consistent with the description by Honda et al. that calcium deposition was not observed until 16 weeks after surgery. 11 In addition, a great advantage of mouse models is the availability of genetically modified variants. The hyperlipidemic and atherosclerotic mutant mouse models 6 , 15 , 16 and several genetic mouse models such as the Notch1‐null mouse model 7 , 8 , 9 have restricted their application. Our model, however, is established in wild type mice. A benefit of its independence from genetic background is greater flexibility to investigate the roles of specific genes, proteins or risk factors in the development of CAVS. There is also no need to crossbreed transgenic mice, which saves an enormous amount of time, and reduces costs and the number of experimental animals needed. In summary, we have established a model in wild type mice that demonstrates the key functional and histopathological characteristics seen in human CAVS cases, with the additional advantages of being less time consuming and more affordable to establish, with a higher triggering yield, meanwhile, importantly, also being more ecological, thereby facilitating studies of CAVS on a large scale.

Over the years, it has been recognized that CAVS is a complex disease associated with wear‐and‐tear of the leaflets, inflammation, lipid infiltration, and osteoblastic transition of valve interstitial cells (VICs). 17 It is now generally accepted that VICs play a critical role in the pathogenesis of CAVS. Our approach was based on the hypothesis that osteoblastic transition and synthesis of collagen of VICs could be induced by disrupting calcium‐phosphate homeostasis and mechanical injury. Disrupting calcium‐phosphate homeostasis is often observed in patients with chronic kidney disease, and secondary hyperparathyroidism, which induce a cardiovascular calcification‐prone state. 18 Patients with disrupted calcium‐phosphate homeostasis have a high incidence of calcific aortic valve disease and often show a rapid progression into CAVS. 1 , 19 In ex vivo and in vitro studies, high calcium and phosphates models have been explored to induce calcification of valves and VICs. 20 , 21 Our model emulates the calcified valve progression under conditions akin to disrupting calcium‐phosphate homeostasis in in vivo models, taking advantage of the native internal environment. Remarkable calcium deposition and osteogenic markers were observed at 28 days after model establishment. The model shows an accelerated development into CAVS similar to that of clinical patients. VICs grown in 3D cultures allow the cells to mimic the environment under mechanical stress transferred from the extracellular matrix, 22 which suggests that VICs have high potential to differentiate into myofibroblasts under a stress stimulus. Previous studies in vitro and in vivo also suggested that VICs were induced to undergo osteogenic differentiation when exposed to high mechanical stress, with or without osteogenic medium culture. 23 , 24 , 25 In our model, wire injury is hybridized to advance the understanding role of high mechanical stress in CAVS development. We find that wire injury and vitamin D‐induced disruption of calcium‐phosphate homeostasis complement each other accelerate the organic process to establish a better model of CAVS. Compared to the models established using a simple wire injury or vitamin D alone, incrassation and fibrosis of valve leaflets, strong valve calcification, as well as osteogenic markers elevation were observed at the early stage in the hybrid model. In addition, upregulated CD68 and VCAM1 expression suggested significant inflammation and endothelial damage post model establishment, supporting the response‐to‐tissue‐injury theory. 26 Together, out model is reminiscent of human CAVS development in terms of mechanical stress, disruption of calcium‐phosphate homeostasis, osteoblastic transition of VICs, fibrosis, inflammation and endothelial injury.

There are some limitations of our study. First of all, like all other models developed in mice, it is regrettable that mouse valve leaflets do not have a tri‐layer structure akin to the human leaflets, preventing them from imitating the progress of CAVS in human completely. Secondly, our model was induced in male mice. Further research is required to check the model on female mice and to investigate any sex differences in CAVS. And thirdly, the high mechanically stressed state and mechanical injury are usually caused chronically in patients while our model accelerates this process via wire injury.

In conclusion, we have established a novel hybrid mouse model representing the typical clinical characteristics of CAVS in patients that be induced without genetic intervention, efficiently, at low cost, and robustly. It can provide a fast track way to explore the underlying mechanisms of CAVS and identify efficacy pharmacological targets.

AUTHOR CONTRIBUTIONS

NQ, YW and WH contributed to acquire, analyze, interpret the data. NQ wrote the manuscript. NC, YQ and JC acquired and analyzed the echocardiographic data. JF, DX, HH and SY helped the animal modeling experiments. DZ, HD and DW provided data analysis and discussion. JW and XL conceived and supervised the project and acquired funding. All authors read and approved the final manuscript.

FUNDING INFORMATION

National Natural Science Foundation of China (U22A20267, 82030014, 81770252, 82271606), the Key Program of Major Science and Technology Projects in Zhejiang Province (2021C03097 and 2022C03063), and Binjiang Institute of Zhejiang University (ZY202205SMKY001).

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no competing interests.

ETHICS STATEMENT

The animal experiment protocols were approved by the Institutional Animal Research Committee of Zhejiang University (2021‐NO.009).

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (U22A20267, 82030014, 81770252, 82271606), the Key Program of Major Science and Technology Projects in Zhejiang Province (2021C03097 and 2022C03063), and Binjiang Institute of Zhejiang University (ZY202205SMKY001).
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