The impact of semilunar valve pathology ranging from genetic defects to progressive calcific aroticstenosis is enormous and continues to escalate with the increased survival of these once fatal congenitalabnormalities and the burgeoning aging population. Valve replacement by tissue engineering presents anattractive potential therapeutic, intervention for both children and adults. However, the ultimate success ofthese strategies will be determined by the degree to which they can recapitulate the critical processes ofnormal aortic and pulmonary valve ontogeny. We hypothesize semilunar valve development requires thecarefully orchestrated transformation of a genetically distinct subpopulation of endocardia! cells toprovide unique valvular interstitial cells (VICs) that remodel a defined extracellular matrix andmaintain valve homeostasis in response to degenerative stimuli. Using the genetic reagents developedin our laboratory and the exceptional expertise and resources of the SysCode consortium, we propose todevelop a molecular blueprint of valve development and maturation required for successful tissueengineering. Our strategy is to disrupt a discrete pathway at critical stages of valve morphogenesis toexpose essential homeostatic interactions. Specifically, we propose to: 1) Determine the major regulatorypathways 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 modelof attenuated epitheliahmesenchymal transformation (EMT) and NFATd null mice will be used as model ofaccentuated EMT. Laser Capture Microdissection (LCM) and Imaging Mass Spectrometry (IMS) will beemployed to compare tissue specific gene and protein expression profiles. 2) Define the critical regulatorypathways that characterize valve remodeling and homeostasis in late embryonic and postnatalsemilunar valve. A novel pro-valvar endocardial specific Cre will be used to delete a floxed Tie1 allelewhich results in a hyperplastic valve phenotype. ApoE-/- mice will be used as a model of progressive aorticvalve stenosis. . Mice will be evaluated for alterations in valve leaflet thinning, progression of aorticcalcification. 3) Delineate the essential components of a synthetic matrix required to recapitulate valvedevelopment in vitro. Based; on information obtained we will determine the key components required toinduce EMT and ECM remodeling an in vitro Hyaluronic Acid (HA) Hydrogel culture system. Effectiveness ofmatrix manipulations will be assayed by a)the ability to induce transformation of endocardial cells in whichTGFp signaling has been attenuated by deletion of the Tgfbr2 receptor and b) the ability to recapitulate valveformation via specification and transformation of FACS sorted ES cell derived endocardial cells.
Showing the most recent 10 out of 18 publications
