Soluble and microenvironmental factors that modulate myofibroblastic and calcific differentiation of valvular interstitial cells

Calcific aortic stenosis is the cause of significant morbidity and mortality in the United States. Each year roughly 70,000 people undergo total valve replacement as a result of this disease 1, 2. In spite of this fact, valvular interstitial cells (VICs), the main cell-type of the cardiac leaflet, remain relatively understudied. This heterogeneous population of cells which include fibroblasts, myofibroblasts, and osteoblastic cells, plays an important role in the homeostasis of valve tissue 3-5. This thesis focuses on engineering instructive microenvironments that promote healthy myofibroblastic differentiation or pathological osteoblastic differentiation of VICs using controllable cues. The study of the interplay between myofibroblastic and calcific differentiation of VICs in simplified and controlled microenvironments is critical in developing more complex models of disease evolution and healthy tissue engineered valve substitutes. The overall goal of this thesis research was to study what factors are important in the design of controlled cellular microenvironments that regulate VIC dynamic differentiation between myofibroblastic and pathological osteoblastic phenotypes. First, the influence of exogenously delivered soluble factors, TGF-beta1 and statins, on myofibroblast and calcific differentiation was examined and found to be dependent on the presence and contractility of the myofibroblast marker, alpha smooth muscle actin (alphaSMA) 6. Secondly, the role of culture environment and mechanics, notably substrate stiffness, on VIC differentiation was investigated. Stiffer substrates were identified to be myofibroblast and osteoblastic promoting surfaces while softer substrates limited VIC activity 7. In addition to stiffness, the influence of matrix protein on VIC calcific differentiation was studied. Fibronectin, a ubiquitous extracellular matrix (ECM) protein, was shown to limit VIC calcification while fibrin, a wound healing protein structure accelerated VIC calcification 8. Lastly VIC myofibroblast differentiation was studied in two novel 3D hydrogel platforms based on both proteinaceous gelatin 9 and synthetic (poly (ethylene glycol)) photopolymerized hydrogels 10. In both cases VICs achieved spread morphology and differentiated in response to TGF-beta1. This research provides valuable information for those interested in valvular regenerative medicine and tissue engineering for regulating VIC myofibroblast differentiation, as well as those interested in studying VIC pathobiology and the evolution of valvular disease.;1. Mohler, E.R., et al., Bone formation and inflammation in cardiac valves. Circulation, 103, 1522-1528. (2001). 2. Shavelle, D.M., Otto, C.M., Cardiology - Chap. 9 Aortic Stenosis. 2 ed. H.C. Crawford, DiMarco, J.P., Paulus W.J. 2004: Harcourt International. 1660. 3. Filip, D.A., A. Radu, and M. Simionescu, Interstitial-Cells of the Heart-Valves Possess Characteristics Similar to Smooth-Muscle Cells. Circulation Research, 59, 310-320. (1986). 4. Messier, R.H., et al., Dual Structural and Functional Phenotypes of the Porcine Aortic-Valve Interstitial Population - Characteristics of the Leaflet Myofibroblast. Journal of Surgical Research, 57, 1-21. (1994). 5. Mohler, E.R., et al., Identification and characterization of calcifying valve cells from human and canine aortic valves. Journal Of Heart Valve Disease, 8, 254-260. (1999). 6. Benton, J.A., et al., Statins block calcific nodule formation of valvular interstitial cells by inhibiting alpha-smooth mucle actin expression. Arteriosclerosis, Thrombosis, and Vascular Biology, In Review. (2009). 7. Kloxin, A.M., J.A. Benton, and K.S. Anseth, In situ elasticity modulation with dynamic substrates directs cell phenotype. Proceedings Of The National Academy Of Sciences Of The United States Of America, In Review. (2009). 8. Benton, J.A., H.B. Kern, and K.S. Anseth, Substrate Properties Influence Calcification in Valvular Interstitial Cell Culture. Journal Of Heart Valve Disease, 17, 689-699. (2008). 9. Benton, J.A., et al., Photocrosslinking of gelatin macromers to synthesize porous hydrogels that promote valvular interstitial cell function. Tissue Engineering, In Press. (2009). 10. Benton, J.A., B.D. Fairbanks, and K.S. Anseth, Characterization of Valvular Interstitial Cell Function in Three Dimensional Matrix Metalloproteinase Degradable PEG Hydrogels. Biomaterials, In Review. (2009).