Neovascularization is an understudied aspect of calcific aortic valve disease (CAVD). Within diseased valves, cells along the neovessels’ periphery stain for pericyte markers, but it is unclear whether valvular interstitial cells (VICs) can demonstrate a pericyte-like phenotype. This investigation examined the perivascular potential of VICs to regulate valve endothelial cell (VEC) organization and explored the role of Angiopoeitin1-Tie2 signaling in this process. Porcine VECs and VICs were fluorescently tracked and co-cultured in Matrigel over 7 days. VICs regulated early VEC network organization in a ROCK-dependent manner, then guided later VEC network contraction through chemoattraction. Unlike vascular control cells, the valve cell cultures ultimately formed invasive spheroids with 3D angiogenic-like sprouts. VECs co-cultured with VICs displayed significantly more invasion than VECs alone; with VICs generally leading and wrapping around VEC invasive sprouts. Lastly, Angiopoietin1-Tie2 signaling was found to regulate valve cell organization during VEC/VIC spheroid formation and invasion. VICs demonstrated pericyte-like behaviors toward VECs throughout sustained co-culture. The change from a vasculogenic network to an invasive sprouting spheroid suggests that both cell types undergo phenotypic changes during long-term culture in the model angiogenic environment. Valve cells organizing into spheroids and undergoing 3D invasion of Matrigel demonstrated several typical angiogenic-like phenotypes dependent on basal levels of Angiopoeitin1-Tie2 signaling and ROCK activation. These results suggest that the ectopic sustained angiogenic environment during the early stages of valve disease promotes organized activity by both VECs and VICs, contributing to neovessel formation and the progression of CAVD.
This is a preview of subscription content, log in to check access.
Buy single article
Instant unlimited access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
RAC-alpha serine/threonine-protein kinase
Calcific aortic valve disease
Delta like ligand four
Endothelial to mesenchymal transformation
Lagrangian corrected forward migration index
Mouse cardiac endothelial cells
Rho-associated protein kinase
Valve endothelial cell
Valvular interstitial cell
Arevalos, C. A., A. T. Walborn, A. A. Rupert, J. M. Berg, E. L. Godfrey, J. M. V. Nguyen, and K. J. Grande-Allen. Regulation of valve endothelial cell vasculogenic network architectures with ROCK and Rac inhibitors. Microvasc. Res. 98C:108–118, 2015.
Armstrong, E. J., and J. Bischoff. Heart valve development: Endothelial cell signaling and differentiation. Circ. Res. 95:459–470, 2004.
Arnaoutova, I., and H. K. Kleinman. In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. Nat. Protoc. 5:628–635, 2010.
Balaoing, L. R., A. D. Post, H. Liu, K. T. Minn, and K. J. Grande-Allen. Age-related changes in aortic valve hemostatic protein regulation. Arterioscler. Thromb. Vasc. Biol. 34:72–80, 2014.
Bosse, K., C. P. Hans, N. Zhao, S. N. Koenig, N. Huang, A. Guggilam, S. LaHaye, G. Tao, P. A. Lucchesi, J. Lincoln, B. Lilly, and V. Garg. Endothelial nitric oxide signaling regulates Notch1 in aortic valve disease. J. Mol. Cell. Cardiol. 60:27–35, 2013.
Bryan, B. A., and P. A. D’Amore. Pericyte isolation and use in endothelial/pericyte coculture models. Methods Enzymol. 443:315–331, 2008.
Chalajour, F., H. Treede, A. Ebrahimnejad, H. Lauke, H. Reichenspurner, and S. Ergun. Angiogenic activation of valvular endothelial cells in aortic valve stenosis. Exp. Cell Res. 298:455–464, 2004.
Fernández-Pisonero, I., J. López, E. Onecha, A. I. Dueñas, P. Maeso, M. S. Crespo, J. A. S. Román, and C. García-Rodríguez. Synergy between sphingosine 1-phosphate and lipopolysaccharide signaling promotes an inflammatory, angiogenic and osteogenic response in human aortic valve interstitial cells. PLoS One 9:e109081, 2014.
Gould, S. T., E. E. Matherly, J. N. Smith, D. D. Heistad, and K. S. Anseth. The role of valvular endothelial cell paracrine signaling and matrix elasticity on valvular interstitial cell activation. Biomaterials 35:3596–3606, 2014.
Gu, X., and K. S. Masters. Role of the Rho pathway in regulating valvular interstitial cell phenotype and nodule formation. Am. J. Physiol. Heart Circ. Physiol. 300:H448–H458, 2011.
Hakuno, D., N. Kimura, and M. Yoshioka. Periostin advances atherosclerotic and rheumatic cardiac valve degeneration by inducing angiogenesis and MMP production in humans and rodents. J. Clin. Invest. 120(7):2292–2306, 2010.
Hakuno, D., N. Kimura, M. Yoshioka, and K. Fukuda. Molecular mechanisms underlying the onset of degenerative aortic valve disease. J. Mol. Med. 87:17–24, 2009.
Hellstrom, M., L.-K. Phng, J. J. Hofmann, E. Wallgard, L. Coultas, P. Lindblom, J. Alva, A.-K. Nilsson, L. Karlsson, N. Gaiano, K. Yoon, J. Rossant, M. L. Iruela-Arispe, M. Kalen, H. Gerhardt, and C. Betsholtz. Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature 445:776–780, 2007.
Hjortnaes, J., K. Shapero, C. Goettsch, J. D. Hutcheson, J. Keegan, J. Kluin, J. E. Mayer, J. Bischoff, and E. Aikawa. Valvular interstitial cells suppress calcification of valvular endothelial cells. Atherosclerosis 242:251–260, 2015.
Jakobsson, L., C. A. Franco, K. Bentley, R. T. Collins, B. Ponsioen, I. M. Aspalter, I. Rosewell, M. Busse, G. Thurston, A. Medvinsky, S. Schulte-Merker, and H. Gerhardt. Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting. Nat. Cell Biol. 12:943–953, 2010.
Lukasz, A., P. Kümpers, and S. David. Role of angiopoietin/tie2 in critical illness: promising biomarker, disease mediator, and therapeutic target? Scientifica 160174:2012, 2012.
Martins, M., S. Warren, C. Kimberley, A. Margineanu, P. Peschard, A. McCarthy, M. Yeo, C. J. Marshall, C. Dunsby, P. M. W. French, and M. Katan. Activity of PLCε contributes to chemotaxis of fibroblasts towards PDGF. J. Cell Sci. 125:5758–5769, 2012.
Mazzone, A., M. C. Epistolato, R. De Caterina, S. Storti, S. Vittorini, S. Sbrana, J. Gianetti, S. Bevilacqua, M. Glauber, A. Biagini, and P. Tanganelli. Neoangiogenesis, T-lymphocyte infiltration, and heat shock protein-60 are biological hallmarks of an immunomediated inflammatory process in end-stage calcified aortic valve stenosis. J. Am. Coll. Cardiol. 43:1670–1676, 2004.
Melero-Martin, J. M., Z. A. Khan, A. Picard, X. Wu, S. Paruchuri, and J. Bischoff. In vivo vasculogenic potential of human blood-derived endothelial progenitor cells. Blood 109:4761–4768, 2007.
Mohler, E. R., F. Gannon, C. Reynolds, R. Zimmerman, M. G. Keane, and F. S. Kaplan. Bone formation and inflammation in cardiac valves. Circulation 103:1522–1528, 2001.
Nguyen, D.-H. T., S. C. Stapleton, M. T. Yang, S. S. Cha, C. K. Choi, P. A. Galie, and C. S. Chen. Biomimetic model to reconstitute angiogenic sprouting morphogenesis in vitro. Proc. Natl. Acad. Sci. USA 110:6712–6717, 2013.
Otrock, Z. K. Z., R. A. R. Mahfouz, J. A. Makarem, and A. I. Shamseddine. Understanding the biology of angiogenesis: Review of the most important molecular mechanisms. Blood Cells Mol. 39:212–220, 2007.
Oubaha, M., M. I. Lin, Y. Margaron, D. Filion, E. N. Price, L. I. Zon, J.-F. Côté, and J.-P. Gratton. Formation of a PKCζ/β-catenin complex in endothelial cells promotes angiopoietin-1-induced collective directional migration and angiogenic sprouting. Blood 120:3371–3381, 2012.
Paranya, G., S. Vineberg, E. L. Dvorin, S. Kaushal, S. J. Roth, E. Rabkin, F. J. Schoen, and J. Bischoff. Aortic valve endothelial cells undergo transforming growth factor-beta-mediated and non-transforming growth factor-beta-mediated transdifferentiation in vitro. Am. J. Pathol. 159:1335–1343, 2001.
Petrie, R. J., A. D. Doyle, and K. M. Yamada. Random versus directionally persistent cell migration. Nat. Rev. Mol. Cell Biol. 10:538–549, 2009.
Sakabe, M., K. Ikeda, K. Nakatani, N. Kawada, K. Imanaka-Yoshida, T. Yoshida, T. Yamagishi, and Y. Nakajima. Rho kinases regulate endothelial invasion and migration during valvuloseptal endocardial cushion tissue formation. Dev. Dyn. 235:94–104, 2006.
Shapero, K., J. Wylie-Sears, R. A. Levine, J. E. Mayer, and J. Bischoff. Reciprocal interactions between mitral valve endothelial and interstitial cells reduce endothelial-to-mesenchymal transition and myofibroblastic activation. J. Mol. Cell. Cardiol. 80:175–185, 2015.
Shworak, N. W. Angiogenic modulators in valve development and disease: does valvular disease recapitulate developmental signaling pathways? Curr. Opin. Cardiol. 19:140–146, 2004.
Stankunas, K., G. K. Ma, F. J. Kuhnert, C. J. Kuo, and C.-P. Chang. VEGF signaling has distinct spatiotemporal roles during heart valve development. Dev. Biol. 347:325–336, 2010.
Syväranta, S., S. Helske, M. Laine, J. Lappalainen, M. Kupari, and M. I. Mäyränpää. K. a Lindstedt, and P. T. Kovanen. Vascular endothelial growth factor-secreting mast cells and myofibroblasts: a novel self-perpetuating angiogenic pathway in aortic valve stenosis. Arterioscler. Thromb. Vasc. Biol. 30:1220–1227, 2010.
Tseng, H., L. R. Balaoing, B. Grigoryan, R. M. Raphael, T. C. Killian, G. R. Souza, and K. J. Grande-Allen. A three-dimensional co-culture model of the aortic valve using magnetic levitation. Acta Biomater. 10:173–182, 2014.
Ucuzian, A. A., A. A. Gassman, A. T. East, and H. P. Greisler. Molecular mediators of angiogenesis. J. Burn Care Res. 31:158–175, 2010.
van Meurs, M., P. Kümpers, J. J. M. Ligtenberg, J. H. J. M. Meertens, G. Molema, and J. G. Zijlstra. Bench-to-bedside review: Angiopoietin signalling in critical illness—a future target? Crit. Care 13:207, 2009.
Walker, G. A., K. S. Masters, D. N. Shah, K. S. Anseth, and L. A. Leinwand. Valvular myofibroblast activation by transforming growth factor-beta: implications for pathological extracellular matrix remodeling in heart valve disease. Circ. Res. 95:253–260, 2004.
Wang, H., B. Sridhar, L. A. Leinwand, and K. S. Anseth. Characterization of cell subpopulations expressing progenitor cell markers in porcine cardiac valves. PLoS One 8:e69667, 2013.
Welch-Reardon, K. M., N. Wu, and C. C. W. Hughes. A role for partial endothelial-mesenchymal transitions in angiogenesis? Arterioscler. Thromb. Vasc. Biol. 35:303–308, 2014.
Witt, W., A. Jannasch, D. Burkhard, T. Christ, U. Ravens, C. Brunssen, A. Leuner, H. Morawietz, K. Matschke, and T. Waldow. Sphingosine-1-phosphate induces contraction of valvular interstitial cells from porcine aortic valves. Cardiovasc. Res. 93:490–497, 2012.
Yoshioka, M., S. Yuasa, K. Matsumura, K. Kimura, T. Shiomi, N. Kimura, C. Shukunami, Y. Okada, M. Mukai, H. Shin, R. Yozu, M. Sata, S. Ogawa, Y. Hiraki, and K. Fukuda. Chondromodulin-I maintains cardiac valvular function by preventing angiogenesis. Nat. Med. 12:1151–1159, 2006.
The authors thank Jennifer Connell, PhD, for editorial assistance and Cindy Farach-Carson, PhD, for use of her microscopy equipment.
Funding was provided by AHA 11IRG5550052 and an NSF Graduate Research Fellowship to C.A.A.
Conflict of interest
The authors have nothing to disclose.
Associate Editor Aleksander S. Popel oversaw the review of this article.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Figure S1: In order to compare the longer term co-culture of VICs and VECs to a known endothelial-pericyte co-culture, MCECs and 10T1/2 cells (to serve as pericytes) were co-cultured on Matrigel. A) Similar to previous reports of vascular endothelial/pericyte co-cultures26, the MCEC (green) and 10T1/2 (red) cells formed interwoven vasculogenic networks. Scale bar represents 100 µm. B) Quantification of MCEC/10T1/2 network size over time. MCEC-only cultures formed nascent networks at 7 hours, with a structure similar to VECs alone23. By 24 hours, the MCEC networks had consolidated with smooth node-to-tubule transitions. By 48 hours, the MCEC-only network structure had regressed substantially. Co-culturing with the 10T1/2 cells maintained network stability over this time period as demonstrated by the slowed network size decrease over time. * represents p-value <0.05. (PDF 4131 kb)
Figure S3: VEC (green)/VIC (red) are seen during network contraction into spheroids during the time period 14-19 hours after seeding onto Matrigel. During this time period, a subpopulation of VICs can be appreciated for influencing VECs organization during network collapse. Scale bar represents 100 µm. (WMV 4042 kb)
Figure S5: Initiation of angiogenic-like sprouts from VEVIS core 30 to 36 hours post seeding. As shown in the movie, VICs (red) are the first cell type to change into an invasive phenotype and begin sprouting into the Matrigel. VECs (green) can be appreciated to begin following behind the leading VICs. Scale bar represents 25 µm. (MP4 742 kb)
Figure S7: 140 hours after seeding, VECs(green) and VICs organize into large, complex, highly branched, interconnected, and invasive sprouts from the core of the spheroid. It is noteworthy the dynamic movement of the VECs along the sprouts and inside the spheroid. Scale bar represents 100 µm. (MP4 3290 kb)
Figure S8: Higher magnification of the tip of the sprouts from supplemental Figure 7. Further appreciation of the dynamic movement of the VECs (red) and VICs along the sprout is possible in this video as different cells take turns taking the role of tip or stalk cell as is seen in vascular angiogenesis. Scale bar represents 100 µm. (MP4 6400 kb)
Figure S9: An example of a VIC (red) demonstrating a LaFMI > 0.17, a chemoattractive effect on local VECs (green), during early network reorganization. Each cell's tracking tracing demonstrates the variability in the directionality of each local VEC that were or were not affected by the VIC in focus. Images were taken from 14 to 19 hours post seeding. Scale bar represents 25 µm. (WMV 942 kb)
Figure S10: After 140 hours in culture, VEVIS sprouts formed complex geometries spreading in 3D. The continued dynamic plasticity of the sprout edge after 7 days of co-culture is demonstrated. Note how VICs (red) can be appreciated wrapping around and sustaining the VEC sprout while other VICs can be found on the leading edge of sprouts. VECs can also be found on the leading edge of sprouts at this time period. Scale bar represent 25 µm. (MP4 2067 kb)
Figure S11: 3D panoramic view along the y-axis of the z-stack reconstructed image of aSMA staining from Figure 5B. Most cells in VEVISs displayed low levels of unpolarized aSMA as opposed to the polarized aSMA found in VICs but not in CD31+ VECs during the network formation at earlier time points (Figure 1C). aSMA (red). F-actin (green). DAPI (blue). (WMV 7942 kb)
About this article
Cite this article
Arevalos, C.A., Berg, J.M., Nguyen, J.M.V. et al. Valve Interstitial Cells Act in a Pericyte Manner Promoting Angiogensis and Invasion by Valve Endothelial Cells. Ann Biomed Eng 44, 2707–2723 (2016) doi:10.1007/s10439-016-1567-9
- Aortic valve
- Valve endothelial cell
- Interstitial cell