In vitro models of aortic valve calcification: solidifying a system.

Calcific aortic valve disease (CAVD) affects 25% of people over 65, and the late-stage stenotic state can only be treated with total valve replacement, requiring 85,000 surgeries annually in the US alone (University of Maryland Medical Center, 2013, http://umm.edu/programs/services/heart-center-programs/cardiothoracic-surgery/valve-surgery/facts). As CAVD is an age-related disease, many of the affected patients are unable to undergo the open-chest surgery that is its only current cure. This challenge motivates the elucidation of the mechanisms involved in calcification, with the eventual goal of alternative preventative and therapeutic strategies. There is no sufficient animal model of CAVD, so we turn to potential in vitro models. In general, in vitro models have the advantages of shortened experiment time and better control over multiple variables compared to in vivo models. As with all models, the hypothesis being tested dictates the most important characteristics of the in vivo physiology to recapitulate. Here, we collate the relevant pieces of designing and evaluating aortic valve calcification so that investigators can more effectively draw significant conclusions from their results.

[1]  K. O’Brien Pathogenesis of calcific aortic valve disease: a disease process comes of age (and a good deal more). , 2006, Arteriosclerosis, thrombosis, and vascular biology.

[2]  I Vesely,et al.  Micromechanics of the fibrosa and the ventricularis in aortic valve leaflets. , 1992, Journal of biomechanics.

[3]  R. Levy,et al.  Transforming growth factor-beta1 mechanisms in aortic valve calcification: increased alkaline phosphatase and related events. , 2007, The Annals of thoracic surgery.

[4]  H. Garner,et al.  Inhibitory Role of Notch1 in Calcific Aortic Valve Disease , 2011, PloS one.

[5]  G. A. Murphy,et al.  Inhibition of calcifying nodule formation in cultured porcine aortic valve cells by nitric oxide donors. , 2009, European journal of pharmacology.

[6]  R. Levy,et al.  Progression of aortic valve stenosis: TGF-beta1 is present in calcified aortic valve cusps and promotes aortic valve interstitial cell calcification via apoptosis. , 2003, The Annals of thoracic surgery.

[7]  Craig A. Simmons,et al.  Animal Models of Calcific Aortic Valve Disease , 2011, International journal of inflammation.

[8]  Y. Bossé,et al.  Inflammation Is Associated with the Remodeling of Calcific Aortic Valve Disease , 2012, Inflammation.

[9]  Ulrich Dahmen,et al.  Atomic-resolution imaging with a sub-50-pm electron probe. , 2009, Physical review letters.

[10]  Michael S Sacks,et al.  The effects of cellular contraction on aortic valve leaflet flexural stiffness. , 2006, Journal of biomechanics.

[11]  Ying C. Song,et al.  Mechanisms of bioprosthetic heart valve calcification1 , 2003, Transplantation.

[12]  M. Simionescu,et al.  Calf cardiac valvular endothelial cells in culture: production of glycosaminoglycans, prostacyclin and fibronectin. , 1988, Journal of molecular and cellular cardiology.

[13]  H. Spurgeon,et al.  Calcification in aging canine aortic valve. , 1986, Scanning electron microscopy.

[14]  R. Hammer,et al.  Low density lipoprotein receptor-negative mice expressing human apolipoprotein B-100 develop complex atherosclerotic lesions on a chow diet: no accentuation by apolipoprotein(a). , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[15]  E. Rubin,et al.  Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells , 1992, Cell.

[16]  S. Hagl,et al.  Influence of receptor activator of nuclear factor kappa B on human aortic valve myofibroblasts. , 2005, Experimental and molecular pathology.

[17]  A. Prat,et al.  The LDLR deficient mouse as a model for aortic calcification and quantification by micro-computed tomography. , 2011, Atherosclerosis.

[18]  A. Chait,et al.  Oxidized Low Density Lipoproteins Regulate Synthesis of Monkey Aortic Smooth Muscle Cell Proteoglycans That Have Enhanced Native Low Density Lipoprotein Binding Properties* , 2000, The Journal of Biological Chemistry.

[19]  O. Tawfik,et al.  Induction of calcification in rabbit aortas by high cholesterol diets: roles of calcifiable vesicles in dystrophic calcification. , 2002, Atherosclerosis.

[20]  C. Nathan,et al.  Nitric oxide as a secretory product of mammalian cells , 1992, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[21]  D. Mozaffarian,et al.  Heart disease and stroke statistics--2012 update: a report from the American Heart Association. , 2012, Circulation.

[22]  J. Cleveland,et al.  Pro-osteogenic phenotype of human aortic valve interstitial cells is associated with higher levels of Toll-like receptors 2 and 4 and enhanced expression of bone morphogenetic protein 2. , 2009, Journal of the American College of Cardiology.

[23]  M. Borggrefe,et al.  Aortic valve calcification: basic science to clinical practice , 2008, Heart.

[24]  R. Ross,et al.  ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. , 1994, Arteriosclerosis and thrombosis : a journal of vascular biology.

[25]  Magdi H. Yacoub,et al.  Collagen synthesis by mesenchymal stem cells and aortic valve interstitial cells in response to mechanical stretch. , 2006, Cardiovascular research.

[26]  L. Jonasson,et al.  Inflammatory Characteristics of Stenotic Aortic Valves: A Comparison between Rheumatic and Nonrheumatic Aortic Stenosis , 2013, Cardiology research and practice.

[27]  Craig A. Simmons,et al.  Spatial Heterogeneity of Endothelial Phenotypes Correlates With Side-Specific Vulnerability to Calcification in Normal Porcine Aortic Valves , 2005, Circulation research.

[28]  B. Cwalina,et al.  Supramolecular structure of human aortic valve and pericardial xenograft material: atomic force microscopy study , 2008, Journal of materials science. Materials in medicine.

[29]  S. Motomura,et al.  Tumor Necrosis Factor-α Accelerates the Calcification of Human Aortic Valve Interstitial Cells Obtained from Patients with Calcific Aortic Valve Stenosis via the BMP2-Dlx5 Pathway , 2011, Journal of Pharmacology and Experimental Therapeutics.

[30]  Y. Deshaies,et al.  A high fat/high carbohydrate diet induces aortic valve disease in C57BL/6J mice. , 2006, Journal of the American College of Cardiology.

[31]  Ajit P. Yoganathan,et al.  Aortic Valve: Mechanical Environment and Mechanobiology , 2013, Annals of Biomedical Engineering.

[32]  A. Helbing,et al.  Arsenazo III: an improvement of the routine calcium determination in serum. , 1991, European journal of clinical chemistry and clinical biochemistry : journal of the Forum of European Clinical Chemistry Societies.

[33]  Farshid Guilak,et al.  Viscoelastic properties of the aortic valve interstitial cell. , 2009, Journal of biomechanical engineering.

[34]  Xiaoping Yang,et al.  Bone morphogenic protein 2 induces Runx2 and osteopontin expression in human aortic valve interstitial cells: role of Smad1 and extracellular signal-regulated kinase 1/2. , 2009, The Journal of thoracic and cardiovascular surgery.

[35]  Joseph Chen,et al.  Calcific nodule morphogenesis by heart valve interstitial cells is strain dependent , 2012, Biomechanics and Modeling in Mechanobiology.

[36]  M. Drolet,et al.  Development of aortic valve sclerosis or stenosis in rabbits: role of cholesterol and calcium. , 2008, The Journal of heart valve disease.

[37]  A. Gotlieb,et al.  Cell biology of valvular interstitial cells. , 1996, The Canadian journal of cardiology.

[38]  R O Bonow,et al.  Atorvastatin inhibits calcification and enhances nitric oxide synthase production in the hypercholesterolaemic aortic valve , 2005, Heart.

[39]  H. D. Liggitt,et al.  Comparative anatomy and histology : a mouse and human atlas , 2011 .

[40]  A. Zallone,et al.  Aortic valvular interstitial cells apoptosis and calcification are mediated by TNF-related apoptosis-inducing ligand. , 2013, International journal of cardiology.

[41]  O. Tawfik,et al.  Mechanism of dystrophic calcification in rabbit aortas: temporal and spatial distributions of calcifying vesicles and calcification-related structural proteins. , 2004, Cardiovascular pathology : the official journal of the Society for Cardiovascular Pathology.

[42]  V. Joag,et al.  The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology. , 2007, The American journal of pathology.

[43]  I. Alferiev,et al.  Triglycidylamine crosslinking of porcine aortic valve cusps or bovine pericardium results in improved biocompatibility, biomechanics, and calcification resistance: chemical and biological mechanisms. , 2005, The American journal of pathology.

[44]  R. Bonow,et al.  Human degenerative valve disease is associated with up-regulation of low-density lipoprotein receptor-related protein 5 receptor-mediated bone formation. , 2006, Journal of the American College of Cardiology.

[45]  M. Enriquez-Sarano,et al.  Role of circulating osteogenic progenitor cells in calcific aortic stenosis. , 2012, Journal of the American College of Cardiology.

[46]  M. Yacoub,et al.  Nano-analytical electron microscopy reveals fundamental insights into human cardiovascular tissue calcification. , 2013, Nature materials.

[47]  C M Johnson,et al.  Porcine cardiac valvular subendothelial cells in culture: cell isolation and growth characteristics. , 1987, Journal of molecular and cellular cardiology.

[48]  Javier Bermejo,et al.  Diet-Induced Aortic Valve Disease in Mice Haploinsufficient for the Notch Pathway Effector RBPJK/CSL , 2011, Arteriosclerosis, thrombosis, and vascular biology.

[49]  Z. Al-Aly,et al.  Aortic Msx2-Wnt Calcification Cascade Is Regulated by TNF-&agr;–Dependent Signals in Diabetic Ldlr−/− Mice , 2007, Arteriosclerosis, thrombosis, and vascular biology.

[50]  Emile R. Mohler,et al.  Bone Formation and Inflammation in Cardiac Valves , 2001, Circulation.

[51]  D. Prockop,et al.  An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. , 2004, Analytical biochemistry.

[52]  B. D. de Smet,et al.  The atherosclerotic Yucatan animal model to study the arterial response after balloon angioplasty: the natural history of remodeling. , 1998, Cardiovascular research.

[53]  Kristi S Anseth,et al.  Substrate properties influence calcification in valvular interstitial cell culture. , 2008, The Journal of heart valve disease.

[54]  D. Towler Molecular and cellular aspects of calcific aortic valve disease. , 2013, Circulation research.

[55]  M. Jauhiainen,et al.  High-density lipoproteins (HDL) are present in stenotic aortic valves and may interfere with the mechanisms of valvular calcification. , 2011, Atherosclerosis.

[56]  J. Kappes,et al.  Oxidative Stress Induces Vascular Calcification through Modulation of the Osteogenic Transcription Factor Runx2 by AKT Signaling* , 2008, Journal of Biological Chemistry.

[57]  Su‐Li Cheng,et al.  Msx2 promotes cardiovascular calcification by activating paracrine Wnt signals. , 2005, The Journal of clinical investigation.

[58]  C. Simmons,et al.  Cofilin is a marker of myofibroblast differentiation in cells from porcine aortic cardiac valves. , 2008, American journal of physiology. Heart and circulatory physiology.

[59]  A. Tajik,et al.  Human Aortic Valve Calcification Is Associated With an Osteoblast Phenotype , 2003, Circulation.

[60]  Robert M Nerem,et al.  Valvular endothelial cells regulate the phenotype of interstitial cells in co-culture: effects of steady shear stress. , 2006, Tissue engineering.

[61]  Kristi S Anseth,et al.  Statins Block Calcific Nodule Formation of Valvular Interstitial Cells by Inhibiting α-Smooth Muscle Actin Expression , 2009, Arteriosclerosis, thrombosis, and vascular biology.

[62]  W. David Merryman,et al.  Mechano-potential etiologies of aortic valve disease. , 2010, Journal of Biomechanics.

[63]  Michael S Sacks,et al.  Synergistic effects of cyclic tension and transforming growth factor-beta1 on the aortic valve myofibroblast. , 2007, Cardiovascular pathology : the official journal of the Society for Cardiovascular Pathology.

[64]  S. Hagl,et al.  Inflammatory regulation of extracellular matrix remodeling in calcific aortic valve stenosis. , 2005, Cardiovascular pathology : the official journal of the Society for Cardiovascular Pathology.

[65]  Rosario V. Freeman,et al.  Spectrum of Calcific Aortic Valve Disease: Pathogenesis, Disease Progression, and Treatment Strategies , 2005, Circulation.

[66]  Craig A Simmons,et al.  Cell–Matrix Interactions in the Pathobiology of Calcific Aortic Valve Disease: Critical Roles for Matricellular, Matricrine, and Matrix Mechanics Cues , 2011, Circulation research.

[67]  L. Leinwand,et al.  Valvular Myofibroblast Activation by Transforming Growth Factor-&bgr;: Implications for Pathological Extracellular Matrix Remodeling in Heart Valve Disease , 2004, Circulation research.

[68]  Landulfo Silveira,et al.  Raman spectroscopy for diagnosis of calcification in human heart valves , 2004 .

[69]  Melody A. Swartz,et al.  Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro , 2005, Journal of Cell Science.

[70]  A. Tajik,et al.  Experimental hypercholesterolemia induces apoptosis in the aortic valve. , 2001, The Journal of heart valve disease.

[71]  Joseph Chen,et al.  Cadherin-11 Regulates Cell–Cell Tension Necessary for Calcific Nodule Formation by Valvular Myofibroblasts , 2013, Arteriosclerosis, thrombosis, and vascular biology.

[72]  J. Lincoln,et al.  Reduced Sox9 Function Promotes Heart Valve Calcification Phenotypes In Vivo , 2010, Circulation research.

[73]  J. Rungby,et al.  The von Kossa reaction for calcium deposits: silver lactate staining increases sensitivity and reduces background , 1993, The Histochemical Journal.

[74]  L. Bonewald,et al.  Von Kossa Staining Alone Is Not Sufficient to Confirm that Mineralization In Vitro Represents Bone Formation , 2003, Calcified Tissue International.

[75]  Urs Utzinger,et al.  Microstructural and biomechanical alterations of the human aorta as a function of age and location , 2010, Biomechanics and modeling in mechanobiology.

[76]  Su‐Li Cheng,et al.  Msx2 Promotes Osteogenesis and Suppresses Adipogenic Differentiation of Multipotent Mesenchymal Progenitors* , 2003, Journal of Biological Chemistry.

[77]  F. Schoen,et al.  Human Pulmonary Valve Progenitor Cells Exhibit Endothelial/Mesenchymal Plasticity in Response to Vascular Endothelial Growth Factor-A and Transforming Growth Factor-&bgr;2 , 2006, Circulation research.

[78]  R. Weissleder,et al.  Notch Signaling in Cardiovascular Disease and Calcification , 2008, Current cardiology reviews.

[79]  K. Anseth,et al.  Fibroblast growth factor represses Smad-mediated myofibroblast activation in aortic valvular interstitial cells , 2008, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[80]  J. Cooke Flow, NO, and atherogenesis , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[81]  Magdi H Yacoub,et al.  Side-specific endothelial-dependent regulation of aortic valve calcification: interplay of hemodynamics and nitric oxide signaling. , 2013, The American journal of pathology.

[82]  C. Simmons,et al.  Mechanical stimulation and mitogen-activated protein kinase signaling independently regulate osteogenic differentiation and mineralization by calcifying vascular cells. , 2004, Journal of biomechanics.

[83]  Deck Jd,et al.  Endothelial cell orientation on aortic valve leaflets , 1986 .

[84]  R. Weissleder,et al.  Arterial and aortic valve calcification inversely correlates with osteoporotic bone remodelling: a role for inflammation , 2010, European heart journal.

[85]  J. Heikkilä,et al.  Prevalence of aortic valve abnormalities in the elderly: an echocardiographic study of a random population sample. , 1993, Journal of the American College of Cardiology.

[86]  S. Stock,et al.  Atorvastatin Inhibits Hypercholesterolemia-Induced Calcification in the Aortic Valves via the Lrp5 Receptor Pathway , 2005, Circulation.

[87]  Robert M Nerem,et al.  Porcine aortic valve interstitial cells in three-dimensional culture: comparison of phenotype with aortic smooth muscle cells. , 2004, The Journal of heart valve disease.

[88]  E. Mohler,et al.  Models of Aortic Valve Calcification , 2007, Journal of Investigative Medicine.

[89]  R. Brooks,et al.  Dysregulation of antioxidant mechanisms contributes to increased oxidative stress in calcific aortic valvular stenosis in humans. , 2008, Journal of the American College of Cardiology.

[90]  K. Masters,et al.  Can valvular interstitial cells become true osteoblasts? A side-by-side comparison. , 2011, The Journal of heart valve disease.

[91]  Kristi S Anseth,et al.  Activation of valvular interstitial cells is mediated by transforming growth factor-beta1 interactions with matrix molecules. , 2005, Matrix biology : journal of the International Society for Matrix Biology.

[92]  R. Levy,et al.  Identification and characterization of calcifying valve cells from human and canine aortic valves. , 1999, The Journal of heart valve disease.

[93]  J. Cleveland,et al.  Cross-Talk Between the Toll-Like Receptor 4 and Notch1 Pathways Augments the Inflammatory Response in the Interstitial Cells of Stenotic Human Aortic Valves , 2012, Circulation.

[94]  F. Schoen,et al.  Human Semilunar Cardiac Valve Remodeling by Activated Cells From Fetus to Adult: Implications for Postnatal Adaptation, Pathology, and Tissue Engineering , 2006, Circulation.

[95]  R. Bonow,et al.  Atorvastatin Inhibits Hypercholesterolemia-Induced Cellular Proliferation and Bone Matrix Production in the Rabbit Aortic Valve , 2002, Circulation.

[96]  J. Bavaria,et al.  Antioxidant Enzymes Reduce DNA Damage and Early Activation of Valvular Interstitial Cells in Aortic Valve Sclerosis , 2013, Arteriosclerosis, thrombosis, and vascular biology.

[97]  S. Hagl,et al.  Receptor activator of nuclear factor κB ligand and osteoprotegerin regulate aortic valve calcification , 2004 .

[98]  J. Handschel,et al.  Effects of dexamethasone, ascorbic acid and β-glycerophosphate on the osteogenic differentiation of stem cells in vitro , 2013, Stem Cell Research & Therapy.

[99]  H. Aupperle,et al.  Pathology, protein expression and signaling in myxomatous mitral valve degeneration: comparison of dogs and humans. , 2012, Journal of veterinary cardiology : the official journal of the European Society of Veterinary Cardiology.

[100]  B. Hinz,et al.  Myofibroblast contraction activates latent TGF-β1 from the extracellular matrix , 2007, The Journal of cell biology.

[101]  F. Guilak,et al.  Correlation between heart valve interstitial cell stiffness and transvalvular pressure: implications for collagen biosynthesis. , 2006, American journal of physiology. Heart and circulatory physiology.

[102]  M. Yacoub,et al.  Characterization of Porcine Aortic Valvular Interstitial Cell ‘Calcified’ Nodules , 2012, PloS one.

[103]  E. Mohler,et al.  Role for Circulating Osteogenic Precursor Cells in Aortic Valvular Disease , 2011, Arteriosclerosis, thrombosis, and vascular biology.

[104]  K. Cianflone,et al.  Endoplasmic Reticulum Stress Participates in Aortic Valve Calcification in Hypercholesterolemic Animals , 2013, Arteriosclerosis, thrombosis, and vascular biology.

[105]  Ajit P Yoganathan,et al.  Elevated cyclic stretch induces aortic valve calcification in a bone morphogenic protein-dependent manner. , 2010, The American journal of pathology.

[106]  D. Srivastava,et al.  Mutations in NOTCH1 cause aortic valve disease , 2005, Nature.

[107]  C. Simmons,et al.  Inhibition of Pathological Differentiation of Valvular Interstitial Cells by C-Type Natriuretic Peptide , 2011, Arteriosclerosis, thrombosis, and vascular biology.

[108]  P. Lawford,et al.  Characterization of the calcification of cardiac valve bioprostheses by environmental scanning electron microscopy and vibrational spectroscopy , 2007, Journal of microscopy.

[109]  E. Aikawa,et al.  Molecular Imaging Insights Into Early Inflammatory Stages of Arterial and Aortic Valve Calcification , 2011, Circulation research.

[110]  Walter Slavin,et al.  Atomic absorption spectroscopy , 1968 .

[111]  E. Bassi,et al.  Oxidant Generation Predominates Around Calcifying Foci and Enhances Progression of Aortic Valve Calcification , 2008, Arteriosclerosis, thrombosis, and vascular biology.

[112]  S. Hagl,et al.  Expression of bone sialoprotein and bone morphogenetic protein-2 in calcific aortic stenosis. , 2004, The Journal of heart valve disease.

[113]  Kevin Kit Parker,et al.  Cyclic strain induces dual-mode endothelial-mesenchymal transformation of the cardiac valve , 2011, Proceedings of the National Academy of Sciences.

[114]  Shmuel Einav,et al.  Detection of Microcalcification in Tissue by Raman Spectroscopy , 2011 .

[115]  M. Herregods,et al.  Statins for calcific aortic valve stenosis: into oblivion after SALTIRE and SEAS? An extensive review from bench to bedside. , 2010, Current problems in cardiology.

[116]  H. Baldwin,et al.  Myocardial contraction and hyaluronic acid mechanotransduction in epithelial-to-mesenchymal transformation of endocardial cells. , 2014, Biomaterials.

[117]  T. Komori,et al.  Inhibition of Notch1 signaling by Runx2 during osteoblast differentiation , 2011, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[118]  R. Knuechel,et al.  Three-dimensional tissue structure affects sensitivity of fibroblasts to TGF-β1 , 2003 .

[119]  Magdi H Yacoub,et al.  Expression of smooth muscle cell markers and co-activators in calcified aortic valves. , 2015, European heart journal.

[120]  G. Weinmaster,et al.  Notch1 is essential for postimplantation development in mice. , 1994, Genes & development.

[121]  Kozo Nakamura,et al.  Distinct effects of PPARγ insufficiency on bone marrow cells, osteoblasts, and osteoclastic cells , 2005, Journal of Bone and Mineral Metabolism.

[122]  Kristyn S Masters,et al.  Regulation of valvular interstitial cell calcification by adhesive peptide sequences. , 2010, Journal of biomedical materials research. Part A.

[123]  A. Chester,et al.  Molecular and functional characteristics of heart-valve interstitial cells , 2007, Philosophical Transactions of the Royal Society B: Biological Sciences.

[124]  Joshua D. Hutcheson,et al.  5-HT(2B) antagonism arrests non-canonical TGF-β1-induced valvular myofibroblast differentiation. , 2012, Journal of molecular and cellular cardiology.

[125]  Craig A Simmons,et al.  Calcification by Valve Interstitial Cells Is Regulated by the Stiffness of the Extracellular Matrix , 2009, Arteriosclerosis, thrombosis, and vascular biology.

[126]  H. Baldwin,et al.  A novel technique for quantifying mouse heart valve leaflet stiffness with atomic force microscopy. , 2012, The Journal of heart valve disease.

[127]  Bagrat Grigoryan,et al.  A three-dimensional co-culture model of the aortic valve using magnetic levitation. , 2014, Acta biomaterialia.

[128]  A. Gotlieb,et al.  The progression of calcific aortic valve disease through injury, cell dysfunction, and disruptive biologic and physical force feedback loops. , 2013, Cardiovascular pathology : the official journal of the Society for Cardiovascular Pathology.

[129]  G. Gronowicz,et al.  In vitro mineralization of fetal rat parietal bones in defined serum‐free medium: Effect of β‐glycerol phosphate , 1989, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[130]  F. Clubb,et al.  Swine as Models in Biomedical Research and Toxicology Testing , 2012, Veterinary pathology.

[131]  J. Fisher,et al.  Factors influencing the oxygen consumption rate of aortic valve interstitial cells: application to tissue engineering. , 2009, Tissue engineering. Part C, Methods.