The impact of semilunar valve pathology ranging from genetic defects to progressive calcific arotic stenosis is enormous and continues to escalate with the increased survival of these once fatal congenital abnormalities and the burgeoning aging population. Valve replacement by tissue engineering presents an attractive potential therapeutic, intervention for both children and adults. However, the ultimate success of these strategies will be determined by the degree to which they can recapitulate the critical processes of normal aortic and pulmonary valve ontogeny. We hypothesize semilunar valve development requires the carefully orchestrated transformation of a genetically distinct subpopulation of endocardia! cells to provide unique valvular interstitial cells (VICs) that remodel a defined extracellular matrix and maintain valve homeostasis in response to degenerative stimuli. Using the genetic reagents developed in our laboratory and the exceptional expertise and resources of the SysCode consortium, we propose to develop a molecular blueprint of valve development and maturation required for successful tissue engineering. Our strategy is to disrupt a discrete pathway at critical stages of valve morphogenesis to expose essential homeostatic interactions. Specifically, we propose to: 1) Determine the major regulatory pathways that are essential for initiation of valve formation in the outflow tract endocardial cushions (EDC). Endocardial specific deletion of a floxed Alk3 allele will be used to perturb BMP signaling as a model of attenuated epitheliahmesenchymal transformation (EMT) and NFATd null mice will be used as model of accentuated EMT. Laser Capture Microdissection (LCM) and Imaging Mass Spectrometry (IMS) will be employed to compare tissue specific gene and protein expression profiles. 2) Define the critical regulatory pathways that characterize valve remodeling and homeostasis in late embryonic and postnatal semilunar valve. A novel pro-valvar endocardial specific Cre will be used to delete a floxed Tie1 allele which results in a hyperplastic valve phenotype. ApoE-/- mice will be used as a model of progressive aortic valve stenosis. . Mice will be evaluated for alterations in valve leaflet thinning, progression of aortic calcification. 3) Delineate the essential components of a synthetic matrix required to recapitulate valve development in vitro. Based;on information obtained we will determine the key components required to induce EMT and ECM remodeling an in vitro Hyaluronic Acid (HA) Hydrogel culture system. Effectiveness of matrix manipulations will be assayed by a)the ability to induce transformation of endocardial cells in which TGF? signaling has been attenuated by deletion of the Tgfbr2 receptor and b) the ability to recapitulate valve formation via specification and transformation of FACS sorted ES cell derived endocardial cells.
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