Ciliogenesis and Cilia-Mediated Signaling in Cardiac Valve Development and Disease

Mitral valve prolapse (MVP) and bicuspid aortic valve (BAV) are serious heart conditions that affect a combined 3-4% of the world population. These diseases carry high risk for secondary complications including arrhythmia, blood regurgitation, and sudden cardiac death. Often, valve diseases are heritable and have complex origins that are not fully understood. The lack of knowledge about valve disease causation likely contributes to the absence of non-surgical treatment options for this patient population. Recent work by our lab and others has demonstrated a link between defects in primary cilia and the development of heart valve disease in humans. Primary cilia are singular, non-motile, microtubule-derived, signaling organelles that are ubiquitous to most cell types. In cardiac valves, they are spatially and temporally regulated, appearing primarily on interstitial cells during valvulogenesis. This dissertation presents evidence that ciliogenesis in the valves is dependent on a highly conserved octameric protein trafficking complex known as the exocyst. We demonstrate that defects in exocyst trafficking result in shorter, less prevalent cilia, the development of bicuspid aortic valves and myxomatous mitral valves in mice, and arterial stenosis in zebrafish. We also uncovered links between ciliome variants and BAV in humans through GWAS analysis. Additionally, we present findings that establish Desert Hedgehog (DHH) signaling as a necessary ciliary signaling pathway in valvulogenesis. Through morphometric analysis of murine mitral valves and biochemical in vitro studies with embryonic chicken valve cells, we describe a novel paracrine crosstalk mechanism emanating from the DHH expressing endocardium that influences the production of alpha smooth muscle actin (a-SMA) by the ciliated valve interstitial cells to promote valve remodeling. This newly generated hypothesis will guide future studies and may provide mechanistic insights of ciliopathies in other tissues. This project led to a new understanding of valve development and highlights the importance of the exocyst, primary cilia, and Desert Hedgehog signaling in valve morphogenesis. Genetic defects in any portion of this system result in faulty valve architecture and disease progression. Insights from this work will inform future discoveries into disease origins and progression and potentially lead to the identification of therapeutic targets for cardiac valve diseases

Emerging Feminist Voices on Media and Representation

The work featured in this panel is from students in WS2000, Introduction to Women\u27s Studies. I created an assignment called Choose Your Own Adventure. These projects include: an examination gender in film, and a revised version of the Bechdel Test, sexism and misogyny in gaming culture expressed through a series of comics, a painting on canvas using a variety of materials and techniques representing the control of women\u27s reproductive rights and the damage done to female bodies by patriarchal language and rhetoric, and an analysis of womanism, scripture and Alice Walker\u27s The Color Purple. Each student engaged with issues related to feminism, equality, in a variety of media and these young women CRUSHED IT! Cassie Tenorio, The Bechdel Alternativehttp://filminsim.tumblr.com/post/104463462075/bechdel-alternativeAlyssa Wells, Painting on Canvas Savannah Fulmer, Comics, Gamer Culture and Gende

PDGFRα: Expression and Function during Mitral Valve Morphogenesis

Mitral valve prolapse (MVP) is a common form of valve disease and can lead to serious secondary complications. The recent identification of MVP causal mutations in primary cilia-related genes has prompted the investigation of cilia-mediated mechanisms of disease inception. Here, we investigate the role of platelet-derived growth factor receptor-alpha (PDGFRα), a receptor known to be present on the primary cilium, during valve development using genetically modified mice, biochemical assays, and high-resolution microscopy. While PDGFRα is expressed throughout the ciliated valve interstitium early in development, its expression becomes restricted on the valve endocardium by birth and through adulthood. Conditional ablation of Pdgfra with Nfatc1-enhancer Cre led to significantly enlarged and hypercellular anterior leaflets with disrupted endothelial adhesions, activated ERK1/2, and a dysregulated extracellular matrix. In vitro culture experiments confirmed a role in suppressing ERK1/2 activation while promoting AKT phosphorylation. These data suggest that PDGFRα functions to suppress mesenchymal transformation and disease phenotypes by stabilizing the valve endocardium through an AKT/ERK pathway

Dynamic Expression Profiles of β-Catenin during Murine Cardiac Valve Development

β-catenin has been widely studied in many animal and organ systems across evolution, and gain or loss of function has been linked to a number of human diseases. Yet fundamental knowledge regarding its protein expression and localization remains poorly described. Thus, we sought to define whether there was a temporal and cell-specific regulation of β-catenin activities that correlate with distinct cardiac morphological events. Our findings indicate that activated nuclear β-catenin is primarily evident early in gestation. As development proceeds, nuclear β-catenin is down-regulated and becomes restricted to the membrane in a subset of cardiac progenitor cells. After birth, little β-catenin is detected in the heart. The co-expression of β-catenin with its main transcriptional co-factor, Lef1, revealed that Lef1 and β-catenin expression domains do not extensively overlap in the cardiac valves. These data indicate mutually exclusive roles for Lef1 and β-catenin in most cardiac cell types during development. Additionally, these data indicate diverse functions for β-catenin within the nucleus and membrane depending on cell type and gestational timing. Cardiovascular studies should take into careful consideration both nuclear and membrane β-catenin functions and their potential contributions to cardiac development and disease

Desert Hedgehog-Primary Cilia Cross Talk Shapes Mitral Valve Tissue by Organizing Smooth Muscle Actin

Non-syndromic mitral valve prolapse (MVP) is the most common heart valve disease affecting 2.4% of the population. Recent studies have identified genetic defects in primary cilia as causative to MVP, although the mechanism of their action is currently unknown. Using a series of gene inactivation approaches, we define a paracrine mechanism by which endocardially-expressed Desert Hedgehog (DHH) activates primary cilia signaling on neighboring valve interstitial cells. High-resolution imaging and functional assays show that DHH de-represses smoothened at the primary cilia, resulting in kinase activation of RAC1 through the RAC1-GEF, TIAM1. Activation of this non-canonical hedgehog pathway stimulates α-smooth actin organization and ECM remodeling. Genetic or pharmacological perturbation of this pathway results in enlarged valves that progress to a myxomatous phenotype, similar to valves seen in MVP patients. These data identify a potential molecular origin for MVP as well as establish a paracrine DHH-primary cilium cross-talk mechanism that is likely applicable across developmental tissue types

Mitral Valve Prolapse Induces Regionalized Myocardial Fibrosis

Background Mitral valve prolapse (MVP) is one of the most common forms of cardiac valve disease and affects 2% to 3% of the population. Previous imaging reports have indicated that myocardial fibrosis is common in MVP and described its association with sudden cardiac death. These data combined with evidence for postrepair ventricular dysfunction in surgical patients with MVP support a link between fibrosis and MVP. Methods and Results We performed histopathologic analysis of left ventricular (LV) biopsies from peripapillary regions, inferobasal LV wall and apex on surgical patients with MVP, as well as in a mouse model of human MVP (Dzip1S14R/+). Tension‐dependent molecular pathways were subsequently assessed using both computational modeling and cyclical stretch of primary human cardiac fibroblasts in vitro. Histopathology of LV biopsies revealed regionalized fibrosis in the peripapillary myocardium that correlated with increased macrophages and myofibroblasts. The MVP mouse model exhibited similar regional increases in collagen deposition that progress over time. As observed in the patient biopsies, increased macrophages and myofibroblasts were observed in fibrotic areas within the murine heart. Computational modeling revealed tension‐dependent profibrotic cellular and molecular responses consistent with fibrosis locations related to valve‐induced stress. These simulations also identified mechanosensing primary cilia as involved in profibrotic pathways, which was validated in vitro and in human biopsies. Finally, in vitro stretching of primary human cardiac fibroblasts showed that stretch directly activates profibrotic pathways and increases extracellular matrix protein production. Conclusions The presence of prominent regional LV fibrosis in patients and mice with MVP supports a relationship between MVP and progressive damaging effects on LV structure before overt alterations in cardiac function. The regionalized molecular and cellular changes suggest a reactive response of the papillary and inferobasal myocardium to increased chordal tension from a prolapsing valve. These studies raise the question whether surgical intervention on patients with MVP should occur earlier than indicated by current guidelines to prevent advanced LV fibrosis and potentially reduce residual risk of LV dysfunction and sudden cardiac death