Heart valves reside in a dynamic mechanical environment experiencing a multitude of forces. These forces have been shown to play a key role in valvular pathophysiology. However, knowledge of proteins involved in valvular mechanobiology is limited to only a few proteins of interest, namely, α-smooth muscle actin, transforming growth factor β, etc. Valvular endothelium mediates valvular homeostasis and controls valvular interstitial cell phenotype transformation. But, how endothelium mediates valvular response to dynamic forces is also unknown. In this study, proteomic analysis of mitral valve anterior leaflets under 10% cyclic radial strain was performed. Endothelium from these samples was removed to test how endothelium mediates mitral valve response to stretch. Results show that stretch downregulated cytoskeletal proteins and proteins involved in energy metabolism such as glycolysis and oxireductase activity. Endothelium removal resulted in downregulation of extracellular matrix and cell-matrix adhesion proteins. Removal of endothelium also resulted in upregulation of translation-related and chaperone proteins. Overall, this high throughput study provides insights into new protein groups that may be involved in mitral valve response to mechanical stretch and loss of endothelium.
Impact statement
This work is important to the field of heart valve pathophysiology as it provides new insights into molecular markers of mechanically induced valvular degeneration as well as the protective role of the valvular endothelium. These discoveries reported here advance our current knowledge of the valvular endothelium and how its removal essentially takes valve leaflets into an environmental shock. In addition, it shows that static conditions represent a mild pathological state for valve leaflets, while 10% cyclic stretch provides valvular cell quiescence. These findings impact the field by informing disease stages and by providing potential new drug targets to reverse or slow down valvular change before it affects cardiac function.
LiuACJoagVRGotliebAI.The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology. Am J Pathol2007;
171:1407–18
2.
ButcherJTNeremRM.Valvular endothelial cells and the mechanoregulation of valvular pathology. Philos Trans R Soc Lond B Biol Sci2007;
362:1445–57
3.
David MerrymanW.Mechano-potential etiologies of aortic valve disease. J Biomech2010;
43:87–92
4.
WangHLeinwandLAAnsethKS.Cardiac valve cells and their microenvironment – insights from in vitro studies. Nat Rev Cardiol2014;
11:715–27
5.
Rabkin-AikawaEFarberMAikawaMSchoenFJ.Dynamic and reversible changes of interstitial cell phenotype during remodeling of cardiac valves. J Heart Valve Dis2004;
13:841–7
6.
RuizJLHutchesonJDAikawaE.Cardiovascular calcification: current controversies and novel concepts. Cardiovasc Pathol2015;
24:207–12
7.
R. Lacerda CMOrtonEC.Evidence of a role for tensile loading in the pathogenesis of mitral valve degeneration. J Clin Exp Cardiolog2012;
1:1–5
8.
LacerdaCMRMacLeaHBKisidayJDOrtonEC.Static and cyclic tensile strain induce myxomatous effector proteins and serotonin in canine mitral valves. J Vet Cardiol2012;
14:223–30
Thayer P, Balachandran K, Rathan S, Yap CH, Arjunon S, Jo H, Yoganathan AP.
The effects of combined cyclic stretch and pressure on the aortic valve interstitial cell phenotype. Ann Biomed Eng2011;
39:1654–67
11.
Sacks MS, Enomoto Y, Graybill JR, Merryman WD, Zeeshan A, Yoganathan AP, Levy RJ, Gorman RC, Gorman JH.
In-vivo dynamic deformation of the mitral valve anterior leaflet. Ann Thorac Surg2006;
82:1369–77
12.
SacksMSYoganathanAP.Heart valve function: a biomechanical perspective. Philos Trans R Soc Lond B Biol Sci2007;
362:1369–91
13.
KunzelmanKSEinsteinDRCochranRP.Fluid-structure interaction models of the mitral valve: function in normal and pathological states. Philos Trans R Soc2007;
362:1393–406
14.
Gould RA, Chin K, Santisakultarm TP, Dropkin A, Richards JM, Schaffer CB, Butcher JT.
Cyclic strain anisotropy regulates valvular interstitial cell phenotype and tissue remodeling in three-dimensional culture. Acta Biomater2012;
8:1710–9
15.
SmithKEMetzlerSAWarnockJN.Cyclic strain inhibits acute pro-inflammatory gene expression in aortic valve interstitial cells. Biomech Model Mechanobiol2010;
9:117–25
16.
Merryman WD, Lukoff HD, Long RA, Engelmayr GC, Hopkins RA, Sacks MS, Sacks MS.
Synergistic effects of cyclic tension and transforming growth factor-beta1 on the aortic valve myofibroblast. Cardiovasc Pathol2007;
16:268–76
17.
GuptaVTsengHLawrenceBDJane Grande-AllenK.Effect of cyclic mechanical strain on glycosaminoglycan and proteoglycan synthesis by heart valve cells. Acta Biomater2009;
5:531–40
18.
FisherCIChenJMerrymanWD.Calcific nodule morphogenesis by heart valve interstitial cells is strain dependent. Biomech Model Mechanobiol2013;
12:5–17
19.
HutchesonJDVenkataramanRBaudenbacherFJDavid MerrymanW.Intracellular Ca 2+ accumulation is strain-dependent and correlates with apoptosis in aortic valve fibroblasts. J Biomech2012;
45:888–94
20.
AliMSWangXLacerdaCMR.A survey of membrane receptor regulation in valvular interstitial cells cultured under mechanical stresses. Exp Cell Res2017;
351:150–6
21.
BalachandranKKonduriSSucoskyPJoHYoganathanAP.An ex vivo study of the biological properties of porcine aortic valves in response to circumferential cyclic stretch. Ann Biomed Eng2006;
34:1655–65
22.
BalachandranKSucoskyPJoHYoganathanAP.Elevated cyclic stretch alters matrix remodeling in aortic valve cusps: implications for degenerative aortic valve disease. Am J Physiol Heart Circ Physiol2009;
296:H756–64
23.
BalachandranKSucoskyPJoHYoganathanAP.Elevated cyclic stretch induces aortic valve calcification in a bone morphogenic protein-dependent manner. Am J Pathol2010;
177:49–57
24.
Pedersen LG, Zhao J, Yang J, Thomsen PD, Gregersen H, Hasenkam JM, Smerup M, Pedersen HD, Olsen LH.
Increased expression of endothelin B receptor in static stretch exposed porcine mitral valve leaflets. Res Vet Sci2007;
82:232–8
25.
MahlerGJFarrarEJButcherJT.Inflammatory cytokines promote mesenchymal transformation in embryonic and adult valve endothelial cells. Arterioscler Thromb Vasc Biol2013;
33:121–30
26.
GouldSTMatherlyEESmithJNHeistadDDAnsethKS.The role of valvular endothelial cell paracrine signaling and matrix elasticity on valvular interstitial cell activation. Biomaterials2014;
35:3596–606
27.
ButcherJTNeremRM.Valvular endothelial cells regulate the phenotype of interstitial cells in co-culture: effects of steady shear stress. Tissue Eng2006;
12:905–15
28.
ShaperoKWylie-SearsJLevineR. AMayerJEBischoffJ.Reciprocal interactions between mitral valve endothelial and interstitial cells reduce endothelial-to-mesenchymal transition and myofibroblastic activation. J Mol Cell Cardiol2015;
80:175–85
29.
Richards J, El-Hamamsy I, Chen S, Sarang Z, Sarathchandra P, Yacoub MH, Chester AH, Butcher JT.
Side-specific endothelial-dependent regulation of aortic valve calcification: interplay of hemodynamics and nitric oxide signaling. Am J Pathol2013;
182:1922–31
30.
Balachandran K, Alford PW, Wylie-Sears J, Goss J a., Grosberg a., Bischoff J, Aikawa E, Levine R a., Parker KK.
Cyclic strain induces dual-mode endothelial-mesenchymal transformation of the cardiac valve. Proc Natl Acad Sci U S A2011;
108:19943–8
31.
Hjortnaes J, Shapero K, Goettsch C, Hutcheson JD, Keegan J, Kluin J, Mayer JE, Bischoff J, Aikawa E.
Valvular interstitial cells suppress calcification of valvular endothelial cells. Atherosclerosis2015;
242:251–60
32.
LeaskRLJainNButanyJ.Endothelium and valvular diseases of the heart. Microsc Res Tech2003;
60:129–37
33.
Kottapalli KR, Zabet-Moghaddam M, Rowland D, Faircloth W, Mirzaei M, Haynes PA, Payton P.
Shotgun label-free quantitative proteomics of water-deficit-stressed midmature peanut (Arachis hypogaea L.) seed. J Proteome Res2013;
12:5048–57
34.
CoxJHeinMYLuberCAParonINagarajNMannM.Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteom2014;
13:2513–26
35.
Saeed AI, Bhagabati NK, Braisted JC, Liang W, Sharov V, Howe EA, Li J, Thiagarajan M, White JA, Quackenbush J.
TM4 microarray software suite. Meth Enzymol2006;
411:134–93
36.
Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J, Simonovic M, Roth A, Santos A, Tsafou KP, Kuhn M, Bork P, Jensen LJ, von Mering C.
STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res2015;
43:D447–52
37.
Szklarczyk D, Morris JH, Cook H, Kuhn M, Wyder S, Simonovic M, Santos A, Doncheva NT, Roth A, Bork P, Jensen LJ, Von Mering C.
The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res2017;
45:D362–8
38.
MiHMuruganujanACasagrandeJTThomasPD.Large-scale gene function analysis with the PANTHER classification system. Nat Protoc2013;
8:1551–66
39.
Mi H, Huang X, Muruganujan A, Tang H, Mills C, Kang D, Thomas PD.
PANTHER version 11: expanded annotation data from gene ontology and reactome pathways, and data analysis tool enhancements. Nucleic Acids Res2017;
45:D183–9
40.
RichardsJMFarrarEJKornreichBGMoseNSButcherJT.The mechanobiology of mitral valve function, degeneration, and repair. J Vet Cardiol2012;
14:47–58
41.
LacerdaCMRDisatianSOrtonEC.Differential protein expression between normal, early-stage, and late-stage myxomatous mitral valves from dogs. Proteomics2009;
3:1422–9
42.
Norris RA, Moreno-Rodriguez R, Wessels A, Merot J, Bruneval P, Chester AH, Yacoub MH, Hagège A, Slaugenhaupt SA, Aikawa E, Schott JJ, Lardeux A, Harris BS, Williams LK, Richards A, Levine RA, Markwald RR.
Expression of the familial cardiac valvular dystrophy gene, filamin-A, during heart morphogenesis. Dev Dyn2010;
239:2118–27
43.
Kyndt F, Gueffet J-P, Probst V, Jaafar P, Legendre A, Le Bouffant F, Toquet C, Roy E, McGregor L, Lynch SA, Newbury-Ecob R, Tran V, Young I, Trochu J-N, Le Marec H, Schott J-J.
Mutations in the gene encoding filamin A as a cause for familial cardiac valvular dystrophy. Circulation2006;
115:40–9
44.
SaulsKDe VlamingAHarrisBS Williams K, Wessels A, Levine RA, Slaugenhaupt SA, Goodwin RL, Pavone LM, Merot J, Schott JJ, Le Tourneau T, Dix T, Jesinkey S, Feng Y, Walsh C, Zhou B, Baldwin S, Markwald RR, Norris RA.
Developmental basis for filamin-A-associated myxomatous mitral valve disease. Cardiovasc Res2012;
96:109–19
45.
FengYWalshCA.The many faces of filamin: a versatile molecular scaffold for cell motility and signalling. Nat Cell Biol2004;
6:1034–8
46.
Duval D, Lardeux A, Le Tourneau T, Norris RA, Markwald RR, Sauzeau V, Probst V, Le Marec H, Levine R, Schott JJ, Merot J.
Valvular dystrophy associated filamin A mutations reveal a new role of its first repeats in small-GTPase regulation. Biochim Biophys Acta2014;
1843:324–34
47.
HartlFUBracherAHayer-HartlM.Molecular chaperones in protein folding and proteostasis. Nature2011;
475:324–32
48.
Lamirault G, Gaborit N, Le Meur N, Chevalier C, Lande G, Demolombe S, Escande D, Nattel S, Léger JJ, Steenman M.
Gene expression profile associated with chronic atrial fibrillation and underlying valvular heart disease in man. J Mol Cell Cardiol2006;
40:173–84
49.
TuTZhouSLiuZLiXLiuQ.Quantitative proteomics of changes in energy metabolism-related proteins in atrial tissue from valvular disease patients with permanent atrial fibrillation. Circ J2014;
78:993–1001
50.
Katz R, Wong ND, Kronmal R, Takasu J, Shavelle DM, Probstfield JL, Bertoni AG, Budoff MJ, O’Brien KD.
Features of the metabolic syndrome and diabetes mellitus as predictors of aortic valve calcification in the multi-ethnic study of atherosclerosis. Circulation2006;
113:2113–9
51.
Briand M, Lemieux I, Dumesnil JG, Mathieu P, Cartier A, Després JP, Arsenault M, Couet J, Pibarot P.
Metabolic syndrome negatively influences disease progression and prognosis in aortic stenosis. J Am Coll Cardiol2006;
47:2229–36
52.
Briand M, Pibarot P, Després J-PP, Voisine P, Dumesnil JG, Dagenais F, Mathieu P.
Metabolic syndrome is associated with faster degeneration of bioprosthetic valves. Circulation2006;
114:512–7
53.
MabryKMPayneSZAnsethKS.Microarray analyses to quantify advantages of 2D and 3D hydrogel culture systems in maintaining the native valvular interstitial cell phenotype. Biomaterials2016;
74:31–41
54.
de Jonge HJM, Fehrmann RSN, de Bont ESJM, Hofstra RMW, Gerbens F, Kamps WA, de Vries EGE, van der Zee AGJ, te Meerman GJ, ter Elst A.
Evidence based selection of housekeeping genes.PLoS One. 2007;
2:e898
55.
GeigerRCTaylorWGlucksbergMRDeanDA.Cyclic stretch-induced reorganization of the cytoskeleton and its role in enhanced gene transfer. Gene Ther2006;
13:725–31
56.
KubitschkeHSchnaussJNnetuKDWarmtEStangeRKaesJ.Actin and microtubule networks contribute differently to cell response for small and large strains. New J Phys2017;
19:093003
57.
Morioka M, Parameswaran H, Naruse K, Kondo M, Sokabe M, Hasegawa Y, Suki B, Ito S.
Microtubule dynamics regulate cyclic stretch-induced cell alignment in human airway smooth muscle cells. PLoS One2011;
6:1–9
58.
WibergCHeinegårdDWenglénCTimplRMörgelinM.Biglycan organizes collagen VI into hexagonal-like networks resembling tissue structures. J Biol Chem2002;
277:49120–6
59.
KalamajskiSOldbergA.The role of small leucine-rich proteoglycans in collagen fibrillogenesis. Matrix Biol2010;
29:248–53
60.
GuptaVWerdenbergJALawrenceBDMendezJSStephensEHGrande-AllenKJ.Reversible secretion of glycosaminoglycans and proteoglycans by cyclically stretched valvular cells in 3D culture. Ann Biomed Eng2008;
36:1092–103
61.
RabkinEAikawaMStoneJRFukumotoYLibbyPSchoenFJ.Activated interstitial myofibroblasts express catabolic enzymes and mediate matrix remodeling in myxomatous heart valves. Circulation2001;
104:2525–32
YáñezMGil-LongoJCampos-ToimilM. Calcium binding proteins. In: Islam MS, ed. Advances in experimental medicine and biology. Vol. 740. ■: Springer, 2012, pp.461–482
64.
RescherUGerkeV.Annexins – unique membrane binding proteins with diverse functions. J Cell Sci2004;
117:2631–9
65.
GerkeVMossSE.Annexins: from structure to function. Physiol Rev2002;
82:331–71
66.
HawkinsTEMerrifieldCJMossSE.Calcium signaling and annexins. Cell Biochem Biophys2000;
33:275–96
67.
LizarbeMABarrasaJIOlmoNGavilanesFTurnayJ.Annexin-phospholipid interactions. Functional implications. Int J Mol Sci2013;
14:2652
68.
Cui L, Rashdan NA, Zhu D, Milne EM, Ajuh P, Milne G, Helfrich MH, Lim K, Prasad S, Lerman DA, Vesey AT, Dweck MR, Jenkins WS, Newby DE, Farquharson C, Macrae VE.
End stage renal disease-induced hypercalcemia may promote aortic valve calcification via Annexin VI enrichment of valve interstitial cell derived-matrix vesicles. J Cell Physiol2017;
232:2985–95
69.
Bakhshian NikAHutchesonJDAikawaE.Extracellular vesicles as mediators of cardiovascular calcification. Front Cardiovasc Med2017;
4:78
70.
KrohnJBHutchesonJDMartínez-MartínezEAikawaE.Extracellular vesicles in cardiovascular calcification: expanding current paradigms. J Physiol2016;
594:2895–903
71.
WalkerGAMastersKSShahDNAnsethKSLeinwandL. A.Valvular myofibroblast activation by transforming growth factor-β: implications for pathological extracellular matrix remodeling in heart valve disease. Circ Res2004;
95:253–60
72.
LiuACGotliebAI.Transforming growth factor-beta regulates in vitro heart valve repair by activated valve interstitial cells. J Pathol2008;
173:1275–85
73.
QuinlanAMTBilliarKL.Investigating the role of substrate stiffness in the persistence of valvular interstitial cell activation. J Biomed Mater Res A2012;
100 A:2474–82
74.
LiCGotliebAI.Transforming growth factor-β regulates the growth of valve interstitial cells in vitro. Am J Pathol2011;
179:1746–55
75.
AliMSDebNWangXRahmanMChristopherGFLacerdaCMR.Correlation between valvular interstitial cell morphology and phenotypes: a novel way to detect activation. Tissue Cell2018;
54:38–46
76.
MillerJDChuYBrooksRMRichenbacherWEPeña-SilvaRHeistadDD.Dysregulation of antioxidant mechanisms contributes to increased oxidative stress in calcific aortic valvular stenosis in humans. J Am Coll Cardiol2008;
52:843–50
77.
Liberman M, Bassi E, Martinatti MK, Lario FC, Wosniak J, Pomerantzeff PMA, Laurindo FRM.
Oxidant generation predominates around calcifying foci and enhances progression of aortic valve calcification. Arterioscler Thromb Vasc Biol2008;
28:463–70
78.
Young EWK, Wheeler AR, Simmons CA, Kallenbach K, Rebe P, Oberbeck A, Herden T, Haverich A, Mertsching H, Kaushal S, Vacanti JP, Schoen FJ, Mayer JE.
Matrix-dependent adhesion of vascular and valvular endothelial cells in microfluidic channels. Lab Chip2007;
7:1759
79.
DavisGESengerDR.Endothelial extracellular matrix: biosynthesis, remodeling, and functions during vascular morphogenesis and neovessel stabilization. Circ Res2005;
97:1093–107
80.
MuiznieksLDKeeleyFW.Molecular assembly and mechanical properties of the extracellular matrix: a fibrous protein perspective. Biochim Biophys Acta2013;
1832:866–75
81.
KieltyCM.Elastic fibres in health and disease. Expert Rev Mol Med2006;
8:1–23
82.
Neptune ER, Frischmeyer PA, Arking DE, Myers L, Bunton TE, Gayraud B, Ramirez F, Sakai LY, Dietz HC.
Dysregulation of TGF-β activation contributes to pathogenesis in Marfan syndrome. Nat Genet2003;
33:407–11
VerstraetenAAlaertsMVan LaerLLoeysB.Marfan syndrome and related disorders: 25 years of gene discovery. Hum Mutat2016;
37:524–31
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