Heart valvular endothelial cells (VECs) are distinct from vascular endothelial cells (ECs), but have an uncertain context within the spectrum of known endothelial phenotypes, including lymphatic ECs (LECs). Profiling the phenotypes of the heart valve surface VECs would facilitate identification of a proper seeding population for tissue-engineered valves, as well as elucidate mechanisms of valvular disease. Porcine VECs and porcine aortic ECs (AECs) were isolated from pig hearts and characterized to assess known EC and LEC markers. A transwell migration assay determined their propensity to migrate toward vascular endothelial growth factor, an angiogenic stimulus, over 24 h. Compared to AECs, Flt-1 was expressed on almost double the percentage of VECs, measured as 74 versus 38%. The expression of angiogenic EC markers CXCR4 and DLL4 was >90% on AECs, whereas VECs showed only 35% CXCR4+ and 47% DLL4+. AECs demonstrated greater migration (71.5 ± 11.0 cells per image field) than the VECs with 30.0 ± 15.3 cells per image field (p = 0.032). In total, 30% of VECs were positive for LYVE1+/Prox1+, while these markers were absent in AECs. In conclusion, the population of cells on the surface of heart valves is heterogeneous, consisting largely of nonangiogenic VECs and a subset of LECs. Previous studies have indicated the presence of LECs within the interior of the valves; however, this is the first study to demonstrate their presence on the surface. Identification of this unique endothelial mixture is a step forward in the development of engineered valve replacements as a uniform EC seeding population may not be the best option to maximize transplant success.

Keywords:
Endothelium, Aortic valve, Lymphatic endothelial cells, Angiogenesis
1.
Balaoing, L.R., A.D. Post, H. Liu, K.T. Minn, K.J. Grande-Allen (2014) Age-related changes in aortic valve hemostatic protein regulation. Arterioscler Thromb Vasc Biol 34: 72-80.
2.
Banerji, S., J. Ni, S.-X. Wang, S. Clasper, J. Su, R. Tammi, M. Jones, D.G. Jackson (1999) LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J Cell Biol 144: 789-801.
3.
Blancas, A.A., L.E. Wong, D.E. Glaser, K.E. McCloskey (2012) Specialized tip/stalk-like and phalanx-like endothelial cells from embryonic stem cells. Stem Cell Dev 22: 1398-1407.
4.
Butcher, J.T., A.M. Penrod, A.J. García, R.M. Nerem (2004) Unique morphology and focal adhesion development of valvular endothelial cells in static and fluid flow environments. Arterioscler Thromb Vasc Biol 24: 1429-1434.
5.
Butcher, J.T., S. Tressel, T. Johnson, D. Turner, G. Sorescu, H. Jo, R.M. Nerem (2006) Transcriptional profiles of valvular and vascular endothelial cells reveal phenotypic differences: influence of shear stress. Arterioscler Thromb Vasc Biol 26: 69-77.
6.
Chalajour, F., H. Treede, A. Ebrahimnejad, H. Lauke, H. Reichenspurner, S. Ergun (2004) Angiogenic activation of valvular endothelial cells in aortic valve stenosis. Exp Cell Res 298: 455-464.
7.
Connell, P.S., A.F. Azimuddin, S.E. Kim, F. Ramirez, M.S. Jackson, S.H. Little, K.J. Grande-Allen (2015) Regurgitation hemodynamics alone cause mitral valve remodeling characteristic of clinical disease states in vitro. Ann Biomed Eng, Epub ahead of print.
8.
Deck, J.D. (1986) Endothelial cell orientation on aortic valve leaflets. Cardiovasc Res 20: 760-767.
9.
El-Hamamsy, I., A.H. Chester, M.H. Yacoub (2010) Cellular regulation of the structure and function of aortic valves. J Adv Res 1: 5-12.
10.
Farrar, E.J., J.T. Butcher (2014) Heterogeneous susceptibility of valve endothelial cells to mesenchymal transformation in response to TNFα. Ann Biomed Eng 42: 149-161.
11.
Gerhardt, H., M. Golding, M. Fruttiger, C. Ruhrberg, A. Lundkvist, A. Abramsson, M. Jeltsch, C. Mitchell, K. Alitalo, D. Shima (2003) VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 161: 1163-1177.
12.
Gille, H., J. Kowalski, B. Li, J. LeCouter, B. Moffat, T.F. Zioncheck, N. Pelletier, N. Ferrara (2001) Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2): a reassessment using novel receptor-specific vascular endothelial growth factor mutants. J Biol Chem 276: 3222-3230.
13.
Hellström, M., L.-K. Phng, J.J. Hofmann, E. Wallgard, L. Coultas, P. Lindblom, J. Alva, A.-K. Nilsson, L. Karlsson, N. Gaiano (2007) Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature 445: 776-780.
14.
Hong, Y.K., N. Harvey, Y.H. Noh, V. Schacht, S. Hirakawa, M. Detmar, G. Oliver (2002) Prox1 is a master control gene in the program specifying lymphatic endothelial cell fate. Dev Dyn 225: 351-357.
15.
Johnson, R.A., T.M. Blake (1966) Lymphatics of the heart. Circulation 33: 137-142.
16.
Kholová, I., G. Dragneva, P. Čermáková, S. Laidinen, N. Kaskenpää, T. Hazes, E. Čermáková, I. Šteiner, S. Ylä-Herttuala (2011) Lymphatic vasculature is increased in heart valves, ischaemic and inflamed hearts and in cholesterol-rich and calcified atherosclerotic lesions. Eur J Clin Invest 41: 487-497.
17.
Kholová, I., G. Dragneva, S. Ylä-Herttuala (2013) The lymphatics in normal and pathological heart valves; in Karunamuni G (ed): The Cardiac Lymphatic System. New York, Springer, pp 63-71.
18.
Krueger, J., D. Liu, K. Scholz, A. Zimmer, Y. Shi, C. Klein, A. Siekmann, S. Schulte-Merker, M. Cudmore, A. Ahmed (2011) Flt1 acts as a negative regulator of tip cell formation and branching morphogenesis in the zebrafish embryo. Development 138: 2111-2120.
19.
Mahler, G.J., E.J. Farrar, J.T. Butcher (2013) Inflammatory cytokines promote mesenchymal transformation in embryonic and adult valve endothelial cells. Arterioscler Thromb Vasc Biol 33: 121-130.
20.
Mäkinen, T., T. Veikkola, S. Mustjoki, T. Karpanen, B. Catimel, E.C. Nice, L. Wise, A. Mercer, H. Kowalski, D. Kerjaschki (2001) Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J 20: 4762-4773.
21.
Mazzone, M., D. Dettori, R. Leite de Oliveira, S. Loges, T. Schmidt, B. Jonckx, Y.-M.Tian, A.A. Lanahan, P. Pollard, C. Ruiz de Almodovar (2009) Heterozygous deficiency of PHD2 restores tumor oxygenation and inhibits metastasis via endothelial normalization. Cell 136: 839-851.
22.
Miller, A. (2011) The grossly invisible and generally ignored lymphatics of the mammalian heart. Med Hypotheses 76: 604-606.
23.
Oliver, G. (2004) Lymphatic vasculature development. Nat Rev Immunol 4: 35-45.
24.
Paranya, G., S. Vineberg, E. Dvorin, S. Kaushal, S.J. Roth, E. Rabkin, F.J. Schoen, J. Bischoff (2001) Aortic valve endothelial cells undergo transforming growth factor-β-mediated and non-transforming growth factor-β-mediated transdifferentiation in vitro. Am J Pathol 159: 1335-1343.
25.
Paruchuri, S., J.-H. Yang, E. Aikawa, J.M. Melero-Martin, Z.A. Khan, S. Loukogeorgakis, F.J. Schoen, J. Bischoff (2006) Human pulmonary valve progenitor cells exhibit endothelial/mesenchymal plasticity in response to vascular endothelial growth factor-A and transforming growth factor-β2. Circ Res 99: 861-869.
26.
Puperi, D.S., L.R. Balaoing, R.W. O'Connell, J.L. West, K.J. Grande-Allen (2015) 3-dimensional spatially organized PEG-based hydrogels for an aortic valve co-culture model. Biomaterials 67: 354-364.
27.
Rath, S., M. Salinas, A.G. Villegas, S. Ramaswamy (2015) Differentiation and distribution of marrow stem cells in flex-flow environments demonstrate support of the valvular phenotype. PloS One 10: e0141802.
28.
Sabine, A., Y. Agalarov, H. Maby-El Hajjami, M. Jaquet, R. Hägerling, C. Pollmann, D. Bebber, A. Pfenniger, N. Miura, O. Dormond (2012) Mechanotransduction, PROX1, and FOXC2 cooperate to control connexin37 and calcineurin during lymphatic-valve formation. Dev Cell 22: 430-445.
29.
Simmons, C.A., G.R. Grant, E. Manduchi, P.F. Davies (2005) Spatial heterogeneity of endothelial phenotypes correlates with side-specific vulnerability to calcification in normal porcine aortic valves. Circ Res 96: 792-799.
30.
Strasser, G.A., J.S. Kaminker, M. Tessier-Lavigne (2010) Microarray analysis of retinal endothelial tip cells identifies CXCR4 as a mediator of tip cell morphology and branching. Blood 115: 5102-5110.
31.
Syväranta, S., S. Helske, J. Lappalainen, M. Kupari, P.T. Kovanen (2012) Lymphangiogenesis in aortic valve stenosis - novel regulatory roles for valvular myofibroblasts and mast cells. Atherosclerosis 221: 366-374.
32.
Wiltz, D., C.A. Arevalos, L.R. Balaoing, A.A. Blancas, M.C. Sapp, X. Zhang, K.J. Grande-Allen (2013) Extracellular matrix organization, structure, and function; in Aikawa E (ed): Calcific Aortic Valve Disease. Rijeka, InTech. http://www.intechopen.com/books/calcific-aortic-valve-disease/extracellular-matrix-organization-structure-and-function.
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2016
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