Journal of Investigative Medicine:
Models of Aortic Valve Calcification
Guerraty, Marie; Mohler, Emile R. III
From the Vascular Medicine Section (M.G., E.R.M.), Cardiovascular Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA.
This work was supported in part by the American Heart Association and a National Research Service Award (National Institutes of Health).
Address correspondence to: Dr. Emile R. Mohler III, Hospital of the University of Pennsylvania, 4th Floor Penn Tower Bldg, 34th and Spruce Streets, Philadelphia, PA 19104; e‐mail: firstname.lastname@example.org.
Aortic valve stenosis is a complex inflammatory process, akin to arterial atherosclerosis, involving lymphocytic infiltrates, macrophages, foam cells, endothelial activation and dysfunction, increased cellularity and extracellular matrix deposition, and lipoprotein accumulation. A clonal population of aortic valve myofibroblasts spontaneously undergoes phenotypic transdifferentiation into osteoblast‐like cells and forms calcific nodules in cell culture. Animal models complement these cell culture models by providing in vivo systems in which to study the complex molecular and cellular interactions that cause aortic valve disease in the native hemodynamic and biochemical environment. Whereas some species, such as swine, can develop spontaneous vascular and valvular atherosclerotic lesions, others, such as rabbits and mice, have not been shown to develop lesions naturally and require an inciting factor, such as hypercholesterolemia. In this article, we review the published cell culture and animal models available to study calcific aortic valve disease.
Aortic valve sclerosis (AVS), defined as leaflet thickening with focal areas of calcification without commissural fusion and without significant left ventricular outflow obstruction, affects over 25% of people over 65 years of age.1,2 Sclerosis can progress to stenosis, causing symptoms of left ventricular outflow obstruction, at which point the only treatments are medical management of symptoms and heart remodeling or valve replacement for severe stenosis. AVS was once believed to be a passive degenerative disease, but recent research has shown that it is actually a complex inflammatory process, akin to arterial atherosclerosis, involving lymphocytic infiltrates, macrophages, foam cells, endothelial activation and dysfunction, increased cellularity and extracellular matrix deposition, and lipoprotein accumulation.3‐6 The risk factors for AVS are similar to those for atherosclerosis and include advanced age, male gender, smoking, hypertension, hypercholesterolemia, and diabetes, as well as raised serum calcium and creatinine.7‐9 In this article, we review the published cell culture and animal models available to study calcific aortic valve disease.
Cell Culture Models
The aortic valve is supple tissue that contains a single layer of endothelial cells that envelop a spongiosa layer made up of loosely organized connective tissue on the aortic side and a ventricularis layer containing elastin on the ventricular side. The interstitial myofibroblast, a cell with many similarities to the smooth muscle cell and fibroblast, resides in the interstitial layer of the valve and is thought to assist in maintaining valve integrity in response to mechanical motion, which occurs approximately 100,000 times per day. The myofibroblast normally secretes collagen and other matrix tissue proteins that support the valve architecture. However, the myofibroblast may undergo transdifferentiation, similar to that observed with smooth muscle cells, and participate in pathologic calcification.
A clonal population of myofibroblasts spontaneously undergoes phenotypic transdifferentiation into osteoblast‐like cells and forms calcific nodules in cell culture (Figure 1). The time frame resulting in nodule formation is dependent on the culture substrate and media. Aortic valve myofibroblasts from dogs, pigs, and sheep develop well‐formed calcified nodules in 2 to 3 weeks when cultured on plastic. Human myofibroblasts also develop calcified nodules but develop later, after 4 weeks of culture. Calcified nodules are seen earlier if myofibroblasts are cultured on collagen. The number of calcified nodules increases when β‐glycerophosphate is added to cell culture media.
The clonal population of animal myofibroblasts that forms calcific nodules shares similar phenotypic traits found in human calcified valves, such as secretion of alkaline phosphatase, and extracelllar matrix proteins, such as osteopontin, bone morphogenetic protein 2, and matrix Gla protein (MGP).10 One protein, transforming growth factor β1 (TGF‐β1), which localizes within interstitial cells, inflammatory cells, and calcific deposits in clinical pathology studies of calcific aortic stenosis cusps, also promotes calcific nodule formation.11
Matrix metalloproteinases (MMPs) are zinc‐ and calcium‐dependent endopeptidases that are involved in tissue remodeling and implicated in aortic valve disease. Tenascin (TN‐C), an extracellular matrix glycoprotein found in developing bone and atherosclerotic plaque, when added to cell culture media resulted in increased myofibroblast MMP‐2 messenger ribonucleic acid expression and MMP‐2 gelatinolytic activity (both pro and active forms) compared with control.12 MMP‐9, also present in calcified aortic valves, is secreted only in myofibroblast cultures that contain TGF‐β1.13
Calcified aortic valves contain cholesterol and by‐products such as 25‐hydroxycholesterol and were found to stimulate the formation of calcific nodules in cell culture.10 3‐Hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitors (statins) are currently being tested in clinical trials to determine if cholesterol reduction can halt the progression of aortic valve disease similar to that seen in coronary and carotid arteries. There is in vitro evidence that supports statin therapy as statin drugs were found to inhibit nodule formation via inhibition of the cholesterol biosynthetic pathway and independent of protein prenylation.14 Interestingly, alkaline phosphatase, an important enzyme in calcification of tissue, was also inhibited by statins. Although cell culture experiments do not recapitulate in vivo conditions, the study of calcification of valve interstitial cells such as myofibroblasts may yield insight into the pathologic process and is a simple method for screening potential therapeutic compounds.
Animal models complement these cell culture models by providing in vivo systems in which to study the complex molecular and cellular interactions that cause aortic valve disease in the native hemodynamic and biochemical environment. Whereas some species, such as swine, can develop spontaneous vascular and valvular atherosclerotic lesions, others, such as rabbits and mice, have not been shown to develop lesions naturally.15,16 Conducting experiments on these spontaneous lesions is both inefficient and costly. It is therefore necessary to incite or accelerate the pathologic process by exposure to known risk factors (male gender, age, hypercholesterolemia, hypercalcemia). Several insults to the aortic valve, including advanced age, certain genetic backgrounds (apolipoprotein E, LDL receptor, MADh6, and MGP knockout mice), certain high‐cholesterol, high‐fat, and high‐carbohydrate diets, or combinations of these, have proven effective as outlined below (Table 1). The following models are useful not only for understanding the pathophysiology and natural progression of AVS, but also for gauging the effects of various therapeutic interventions. They may also serve as model systems in which to investigate AV bioprostheses, though this is beyond the scope of this discussion.
Since we are ultimately interested in understanding the mechanism, progression, and treatment of AVS in humans, previous pathologic studies that characterize AVS lesions in humans serve as the gold standard against which to evaluate animal models. Ideally, the AVS induced in animal models would be similar to that of humans in both pathology and distribution; the natural progression of the disease would mirror that in humans; and the models' response to therapies would approximate that of humans. The pathologies incited in animal models should therefore include the following to some degree: leaflet thickening owing to a complex inflammatory process involving lymphocytic infiltrates, macrophages, foam cells, endothelial activation and dysfunction, increased cellularity and extracellular matrix deposition, and lipoprotein accumulation, as well as calcifications, chondroid or osseous metaplasia, or angiogenesis in more advanced lesions.3‐6,17,18 Similarly, in vivo measures of AVS should parallel those seen in humans and include an increased transvalvular velocity, which suggests an increased transvalvular pressure gradient across the aortic valve or a decrease in the aortic valve area.
Mouse models are perhaps the most widely used animal models to study AVS. Their low cost and easy management, along with the availability of clonal samples and genetically mutated strains, have made them a convenient model, for example, a knockout model of Madh6, which encodes the inhibitory, TGF‐β1 signaling mediator protein SMAD6 and causes valve hyperplasia, as well as cartilaginous metaplasia and advanced ossification of the aortic media.19 Smooth muscle cells and chondrocytes of MGP knockout mice calcify spontaneously, leading to premature calcification and massive calcification of the aortic valve and major arteries.20
Mouse models have also been used to study AVS as an aging phenomenon by allowing mice susceptible to atherosclerotic processes to age until they develop valvular disease. This mirrors the progression of AVS in humans where exposure to a systemic risk factor for extended periods of time leads to pathologic changes. Weiss and colleagues allowed low‐density lipoprotein (LDL) receptor knockout mice with only apoliprotein B‐100 to age for approximately 20 months.21 The mice developed moderate hypercholesterolemia and aortic valve disease and, interestingly, showed signs of left ventricular hypertrophy and a decreased ejection fraction, which are characteristic of the clinical syndrome that usually accompanies advanced AVS.21 This model is particularly interesting since it develops the heart disease and hemodynamic effects of aortic valve stenosis, approximating not only valvular pathology but also the ensuing clinical syndrome.
Apolipoprotein E‐deficient mice also develop an increase in transaortic flow velocity with age indicative of aortic valve stenosis.22 Aikawa and colleagues studied these early lesions and found that the aortic valves thickened, contained macrophages and showed early dysfunction in vivo.23 Furthermore, they identified the presence of early biomarkers of AVS in vivo.
A high‐fat, high‐carbohydrate diet, perhaps more relevant to humans than a purely hypercholesterolemic diet, administered to wild‐type and LDL receptor knockout mice resulted in increased transvalvular velocities, that is, decreased aortic valve area, with leaflet thickening and calcium deposition.24
Hypercholesterolemic Rabbit Models
Rabbits have also been used to study early to advanced aortic valve disease. In general, a hypercholesterolemic diet is administered to promote pathogenesis. A 4‐week hypercholesterolemic diet revealed that LDLs accumulate immediately beneath the endothelium and associate with collagen fibers in the extracellular matrix.25,26 A longer, 12‐week, hypercholesterolemic diet induced lipid‐rich lesions and caused an increase in the number of apoptotic (TUNEL positive) cells in the aortic valve.27 When coupled with vitamin D‐induced hypercalcemia, the dietary insult also led to significant calcium deposition and aortic stenosis, detectable by echocardiography.28 Twenty‐ and 40‐week hypercholesterolemic diets also successfully induced early AVS pathology and revealed osteopontin expression in these early lesions.29 Aortic valves from rabbits maintained on a hypercholesterolemic diet for 1 to 20 weeks revealed that β‐very low‐density lipoprotein endocytosis increased after a few weeks and was greater on the aortic side of the leaflet than on the ventricular side.30
Rabbit models have also proven useful in studying the effect of potential therapies on AVS. Rajamannan and colleagues found that atorvastatin administration mediated the cellular proliferation and expression of osteogenic genes induced by hypercholesterolemia.31
Since they can develop spontaneous vascular and valvular lesions, porcine models have been widely used to research atherosclerosis.15 When subjected to an atherogenic diet, swine display lipid profiles comparable to those of humans, particularly with respect to LDLs,32 and atherosclerotic lesions ranging from fibromuscular plaques to more advanced lesions with lipid deposition, calcification, and cholesterol clefts (Figure 2).33‐35 Furthermore, the pig genome is comparable to that of humans in size and is homologous to humans in both sequence and chromosomal structure, making porcine models particularly useful for genomic studies of the aortic valve. Simmons and colleagues used such a model to study the correlation between phenotypic heterogeneity and regions of susceptibility in normal valvular endothelium.36 Since porcine valves are often used to replace dysfunctional human aortic valves, studying the pathophysiology of AVS in swine is also relevant in its own right.
Models of Congenital Valvular Abnormalities
Patients with congenital bicuspid aortic valves develop aortic valve sclerosis and stenosis at an accelerated rate compared with those with normal, trileaflet valves. Both endothelial nitric oxide synthase and Notch1 knockout mice develop congenital biscuspid valves and could potentially serve as useful models for both the congenital disease and the process and progression of AVS.37,38
We thank Peter Davies, PhD, for his advice and support of this review.
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Key words:: calcification; aortic valve; atherosclerosis
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