Epicardial deletion of Sox9 leads to myxomatous valve degeneration and identifies Cd109 as a novel gene associated with valve development.

Epicardial-derived cells (EPDCs) are involved in the regulation of myocardial growth and coronary vascularization and are critically important for proper development of the atrioventricular (AV) valves. SOX9 is a transcription factor expressed in a variety of epithelial and mesenchymal cells in the developing heart, including EPDCs. To determine the role of SOX9 in epicardial development, an epicardial-specific Sox9 knockout mouse model was generated. Deleting Sox9 from the epicardial cell lineage impairs the ability of EPDCs to invade both the ventricular myocardium and the developing AV valves. After birth, the mitral valves of these mice become myxomatous with associated abnormalities in extracellular matrix organization. This phenotype is reminiscent of that seen in humans with myxomatous mitral valve disease (MVD). An RNA-seq analysis was conducted in an effort to identify genes associated with this myxomatous degeneration. From this experiment, Cd109 was identified as a gene associated with myxomatous valve pathogenesis in this model. Cd109 has never been described in the context of heart development or valve disease. This study highlights the importance of SOX9 in the regulation of epicardial cell invasion-emphasizing the importance of EPDCs in regulating AV valve development and homeostasis-and reports a novel expression profile of Cd109, a gene with previously unknown relevance in heart development.

Role of the Epicardium in the Development of the Atrioventricular Valves and Its Relevance to the Pathogenesis of Myxomatous Valve Disease.

This paper is dedicated to the memory of Dr. Adriana "Adri" Gittenberger-de Groot and in appreciation of her work in the field of developmental cardiovascular biology and the legacy that she has left behind. During her impressive career, Dr. Gittenberger-de Groot studied many aspects of heart development, including aspects of cardiac valve formation and disease and the role of the epicardium in the formation of the heart. In this contribution, we review some of the work on the role of epicardially-derived cells (EPDCs) in the development of the atrioventricular valves and their potential involvement in the pathogenesis of myxomatous valve disease (MVD). We provide an overview of critical events in the development of the atrioventricular junction, discuss the role of the epicardium in these events, and illustrate how interfering with molecular mechanisms that are involved in the epicardial-dependent formation of the atrioventricular junction leads to a number of abnormalities. These abnormalities include defects of the AV valves that resemble those observed in humans that suffer from MVD. The studies demonstrate the importance of the epicardium for the proper formation and maturation of the AV valves and show that the possibility of epicardial-associated developmental defects should be taken into consideration when determining the genetic origin and pathogenesis of MVD.

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.

Primary cilia defects causing mitral valve prolapse.

Mitral valve prolapse (MVP) affects 1 in 40 people and is the most common indication for mitral valve surgery. MVP can cause arrhythmias, heart failure, and sudden cardiac death, and to date, the causes of this disease are poorly understood. We now demonstrate that defects in primary cilia genes and their regulated pathways can cause MVP in familial and sporadic nonsyndromic MVP cases. Our expression studies and genetic ablation experiments confirmed a role for primary cilia in regulating ECM deposition during cardiac development. Loss of primary cilia during development resulted in progressive myxomatous degeneration and profound mitral valve pathology in the adult setting. Analysis of a large family with inherited, autosomal dominant nonsyndromic MVP identified a deleterious missense mutation in a cilia gene, DZIP1 A mouse model harboring this variant confirmed the pathogenicity of this mutation and revealed impaired ciliogenesis during development, which progressed to adult myxomatous valve disease and functional MVP. Relevance of primary cilia in common forms of MVP was tested using pathway enrichment in a large population of patients with MVP and controls from previously generated genome-wide association studies (GWAS), which confirmed the involvement of primary cilia genes in MVP. Together, our studies establish a developmental basis for MVP through altered cilia-dependent regulation of ECM and suggest that defects in primary cilia genes can be causative to disease phenotype in some patients with MVP.

Mesothelium and Malignant Mesothelioma.

The mesothelium is an epithelial structure derived from the embryonic mesoderm. It plays an important role in the development of a number of different organs, including the heart, lungs, and intestines. In this publication, we discuss aspects of the development of the mesothelium, where mesothelial structures can be found, and review molecular and cellular characteristics associated with the mesothelium. Furthermore, we discuss the involvement of the mesothelium in a number of disease conditions, in particular in the pathogenesis of mesotheliomas with an emphasis on malignant pleural mesothelioma (MPM)-a primary cancer developing in the pleural cavity.

A Novel Mouse Model for Cilia-Associated Cardiovascular Anomalies with a High Penetrance of Total Anomalous Pulmonary Venous Return.

Primary cilia are small organelles projecting from the cell surface of many cell types. They play a crucial role in the regulation of various signaling pathway. In this study, we investigated the importance of cilia for heart development by conditionally deleting intraflagellar transport protein Ift88 using the col3.6-cre mouse. Analysis of col3.6;Ift88 offspring showed a wide spectrum of cardiovascular defects including double outlet right ventricle and atrioventricular septal defects. In addition, we found that in the majority of specimens the pulmonary veins did not properly connect to the developing left atrium. The abnormal connections found resemble those seen in patients with total anomalous pulmonary venous return. Analysis of mutant hearts at early stages of development revealed abnormal development of the dorsal mesocardium, a second heart field-derived structure at the venous pole intrinsically related to the development of the pulmonary veins. Data presented support a crucial role for primary cilia in outflow tract development and atrioventricular septation and their significance for the formation of the second heart field-derived tissues at the venous pole including the dorsal mesocardium. Furthermore, the results of this study indicate that proper formation of the dorsal mesocardium is critically important for the development of the pulmonary veins. Anat Rec, 302:136-145, 2019. © 2018 Wiley Periodicals, Inc.

A role for primary cilia in aortic valve development and disease.

BACKGROUND: Bicuspid aortic valve (BAV) disease is the most common congenital heart defect, affecting 0.5-1.2% of the population and causing significant morbidity and mortality. Only a few genes have been identified in pedigrees, and no single gene model explains BAV inheritance, thus supporting a complex genetic network of interacting genes. However, patients with rare syndromic diseases that stem from alterations in the structure and function of primary cilia ("ciliopathies") exhibit BAV as a frequent cardiovascular finding, suggesting primary cilia may factor broadly in disease etiology. RESULTS: Our data are the first to demonstrate that primary cilia are expressed on aortic valve mesenchymal cells during embryonic development and are lost as these cells differentiate into collagen-secreting fibroblastic-like cells. The function of primary cilia was tested by genetically ablating the critical ciliogenic gene Ift88. Loss of Ift88 resulted in abrogation of primary cilia and increased fibrogenic extracellular matrix (ECM) production. Consequentially, stratification of ECM boundaries normally present in the aortic valve were lost and a highly penetrant BAV phenotype was evident at birth. CONCLUSIONS: Our data support cilia as a novel cellular mechanism for restraining ECM production during aortic valve development and broadly implicate these structures in the etiology of BAV disease in humans. Developmental Dynamics 246:625-634, 2017. © 2017 Wiley Periodicals, Inc.

The Dorsal Mesenchymal Protrusion and the Pathogenesis of Atrioventricular Septal Defects.

Congenital heart malformations are the most common type of defects found at birth. About 1% of infants are born with one or more heart defect on a yearly basis. Congenital Heart Disease (CHD) causes more deaths in the first year of life than any other congenital abnormality, and each year, nearly twice as many children die in the United States from CHD as from all forms of childhood cancers combined. Atrioventricular septal defects (AVSD) are congenital heart malformations affecting approximately 1 in 2000 live births. Babies born with an AVSD often require surgical intervention shortly after birth. However, even after successful surgery, these individuals typically have to deal with lifelong complications with the most common being a leaky mitral valve. In recent years the understanding of the molecular etiology and morphological mechanisms associated with the pathogenesis of AVSDs has significantly changed. Specifically, these studies have linked abnormal development of the Dorsal Mesenchymal Protrusion (DMP), a Second Heart Field-derived structure, to the development of this congenital defect. In this review we will be discuss some of the latest insights into the role of the DMP in the normal formation of the atrioventricular septal complex and in the pathogenesis of AVSDs.

Mutations in DCHS1 cause mitral valve prolapse.

Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery. Despite a clear heritable component, the genetic aetiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds), that segregates with MVP in the family. Morpholino knockdown of the zebrafish homologue dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 messenger RNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1(+/-) mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs, as well as in Dchs1(+/-) mouse MVICs, result in altered migration and cellular patterning, supporting these processes as aetiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease.

Alk3 mediated Bmp signaling controls the contribution of epicardially derived cells to the tissues of the atrioventricular junction.

Recent studies using mouse models for cell fate tracing of epicardial derived cells (EPDCs) have demonstrated that at the atrioventricular (AV) junction EPDCs contribute to the mesenchyme of the AV sulcus, the annulus fibrosus, and the parietal leaflets of the AV valves. There is little insight, however, into the mechanisms that govern the contribution of EPDCs to these tissues. While it has been demonstrated that bone morphogenetic protein (Bmp) signaling is required for AV cushion formation, its role in regulating EPDC contribution to the AV junction remains unexplored. To determine the role of Bmp signaling in the contribution of EPDCs to the AV junction, the Bmp receptor activin-like kinase 3 (Alk3; or Bmpr1a) was conditionally deleted in the epicardium and EPDCs using the mWt1/IRES/GFP-Cre (Wt1(Cre)) mouse. Embryonic Wt1(Cre);Alk3(fl/fl) specimens showed a significantly smaller AV sulcus and a severely underdeveloped annulus fibrosus. Electrophysiological analysis of adult Wt1(Cre);Alk3(fl/fl) mice showed, unexpectedly, no ventricular pre-excitation. Cell fate tracing revealed a significant decrease in the number of EPDCs within the parietal leaflets of the AV valves. Postnatal Wt1(Cre);Alk3(fl/fl) specimens showed myxomatous changes in the leaflets of the mitral valve. Together these observations indicate that Alk3 mediated Bmp signaling is important in the cascade of events that regulate the contribution of EPDCs to the AV sulcus, annulus fibrosus, and the parietal leaflets of the AV valves. Furthermore, this study shows that EPDCs do not only play a critical role in early developmental events at the AV junction, but that they also are important in the normal maturation of the AV valves.

Developmental basis for filamin-A-associated myxomatous mitral valve disease.

AIMS: We hypothesized that the structure and function of the mature valves is largely dependent upon how these tissues are built during development, and defects in how the valves are built can lead to the pathological progression of a disease phenotype. Thus, we sought to uncover potential developmental origins and mechanistic underpinnings causal to myxomatous mitral valve disease. We focus on how filamin-A, a cytoskeletal binding protein with strong links to human myxomatous valve disease, can function as a regulatory interface to control proper mitral valve development. METHODS AND RESULTS: Filamin-A-deficient mice exhibit abnormally enlarged mitral valves during foetal life, which progresses to a myxomatous phenotype by 2 months of age. Through expression studies, in silico modelling, 3D morphometry, biochemical studies, and 3D matrix assays, we demonstrate that the inception of the valve disease occurs during foetal life and can be attributed, in part, to a deficiency of interstitial cells to efficiently organize the extracellular matrix (ECM). This ECM organization during foetal valve gestation is due, in part, to molecular interactions between filamin-A, serotonin, and the cross-linking enzyme, transglutaminase-2 (TG2). Pharmacological and genetic perturbations that inhibit serotonin-TG2-filamin-A interactions lead to impaired ECM remodelling and engender progression to a myxomatous valve phenotype. CONCLUSIONS: These findings illustrate a molecular mechanism by which valve interstitial cells, through a serotonin, TG, and filamin-A pathway, regulate matrix organization during foetal valve development. Additionally, these data indicate that disrupting key regulatory interactions during valve development can set the stage for the generation of postnatal myxomatous valve disease.

Altered versican cleavage in ADAMTS5 deficient mice; a novel etiology of myxomatous valve disease.

In fetal valve maturation the mechanisms by which the relatively homogeneous proteoglycan-rich extracellular matrix (ECM) of endocardial cushions is replaced by a specialized and stratified ECM found in mature valves are not understood. Therefore, we reasoned that uncovering proteases critical for 'remodeling' the proteoglycan rich (extracellular matrix) ECM may elucidate novel mechanisms of valve development. We have determined that mice deficient in ADAMTS5, (A Disintegrin-like And Metalloprotease domain with ThromboSpondin-type 1 motifs) which we demonstrated is expressed predominantly by valvular endocardium during cardiac valve maturation, exhibited enlarged valves. ADAMTS5 deficient valves displayed a reduction in cleavage of its substrate versican, a critical cardiac proteoglycan. In vivo reduction of versican, in Adamts5(-/-) mice, achieved through Vcan heterozygosity, substantially rescued the valve anomalies. An increase in BMP2 immunolocalization, Sox9 expression and mesenchymal cell proliferation were observed in Adamts5(-/-) valve mesenchyme and correlated with expansion of the spongiosa (proteoglycan-rich) region in Adamts5(-/-) valve cusps. Furthermore, these data suggest that ECM remodeling via ADAMTS5 is required for endocardial to mesenchymal signaling in late fetal valve development. Although adult Adamts5(-/-) mice are viable they do not recover from developmental valve anomalies and have myxomatous cardiac valves with 100% penetrance. Since the accumulation of proteoglycans is a hallmark of myxomatous valve disease, based on these data we hypothesize that a lack of versican cleavage during fetal valve development may be a potential etiology of adult myxomatous valve disease.

Reduced versican cleavage due to Adamts9 haploinsufficiency is associated with cardiac and aortic anomalies.

Here, we demonstrate that ADAMTS9, a highly conserved versican-degrading protease, is required for correct cardiovascular development and adult homeostasis. Analysis of Adamts9(+/LacZ) adult mice revealed anomalies in the aortic wall, valvulosinus and valve leaflets. Abnormal myocardial projections and 'spongy' myocardium consistent with non-compaction of the left ventricle were also found in Adamts9(+/LacZ) mice. During development, Adamts9 was expressed in derivatives of the Secondary Heart Field, vascular smooth muscle cells in the arterial wall, mesenchymal cells of the valves, and non-myocardial cells of the ventricles, but expression also continued in the adult heart and ascending aorta. Thus, the adult cardiovascular anomalies found in Adamts9(+/LacZ) hearts could result from subtle developmental alterations in extracellular matrix remodeling or defects in adult homeostasis. The valvular and aortic anomalies of Adamts9(+/LacZ) hearts were associated with accumulation of versican and a decrease in cleaved versican relative to WT littermates. These data suggest a potentially important role for ADAMTS9 cleavage of versican, or other, as yet undefined substrates in development and allostasis of cardiovascular extracellular matrix. In addition, these studies identify ADAMTS9 as a potential candidate gene for congenital cardiac anomalies. Mouse models of ADAMTS9 deficiency may be useful to study myxomatous valve degeneration.

Morphology and morphogenesis of atrioventricular septal defect with common atrioventricular junction.

For many years, the lesions now often described as atrioventricular septal defects were considered to represent atrioventricular canal malformations or endocardial cushion defects. It was also long recognized that patients with the so-called ostium primum defect should be included in this category. The phenotypic feature of these hearts is the presence of a common atrioventricular junction, as opposed to separate right and left atrioventricular junctions. The presence of the common atrioventricular junction underscores the associated phenotypic features, such as the presence of a trifoliate left atrioventricular valve, which has no resemblance to a cleft mitral valve; unwedging of the subaortic outflow tract; and disproportion between the inlet and outlet dimensions of the left ventricle. These features are comparable in patients having the so-called partial, intermediate, and complete variants of the malformation. Anatomical differentiation depends on the morphology of the leaflets of the common atrioventricular valve that bridge the ventricular septum. If these bridging leaflets are fused one to the other, then there are dual orifices, rather than a common orifice, within the common atrioventricular junction. The relationships of the bridging leaflets to the septal structures determine the potential for shunting across the atrioventricular septal defect, which can occur at atrial and ventricular levels or exclusively at either atrial or ventricular level. Rarely, the atrioventricular septal defect may close spontaneously. Recent evidence from studies of cardiac development shows that rather than being an endocardial cushion defect, the malformation results from failure of ingrowth into the developing heart from the dorsal mesenchymal tissues.

Elevated vascular endothelial cell growth factor affects mesocardial morphogenesis and inhibits normal heart bending.

Signaling by means of vascular endothelial cell growth factor (VEGF) and its receptors (VEGFRs) is required for cardiovascular development. To examine how VEGF/VEGFR receptor signaling affects early endocardial cell behavior, embryonic quail hearts were subjected to elevated VEGF165 levels (five- to nine-somite stage). Primitive embryonic hearts microinjected with recombinant human (rh)VEGF165 exhibit several distinct malformations compared with hearts in untreated embryos: the endocardial tube is malformed with tortuous cords and folds surrounded by a diminished cardiac jelly space, and the lumens of affected hearts are conspicuously reduced. Furthermore, the embryonic heart fails to loop properly. Inhibition of bending is accompanied by an apparent failure of the dorsal mesocardium to atrophy--an event thought to be necessary for heart bending. Instead of atrophy, VEGF-treated mesocardia exhibit a marked increased in the number of resident endothelial cells. Collectively, the data suggest that the abnormally robust mesocardia in VEGF-treated hearts impede the mechanical deformation required for normal heart bending. We conclude that the excessive VEGF signaling culminates in a physical or biomechanical mechanism that acts over a wide, tissue-level, length scale to cause a severe developmental defect--failure of heart bending.

Late gestational lung hypoplasia in a mouse model of the Smith-Lemli-Opitz syndrome.

BACKGROUND: Normal post-squalene cholesterol biosynthesis is important for mammalian embryonic development. Neonatal mice lacking functional dehydrocholesterol Delta7-reductase (Dhcr7), a model for the human disease of Smith-Lemli-Opitz syndrome, die within 24 hours of birth. Although they have a number of biochemical and structural abnormalities, one cause of death is from apparent respiratory failure due to developmental pulmonary abnormalities. RESULTS: In this study, we characterized further the role of cholesterol deficiency in lung development of these mice. Significant growth retardation, beginning at E14.5 through E16.5, was observed in Dhcr7-/- embryos. Normal lobation but smaller lungs with a significant decrease in lung-to-body weight ratio was noted in Dhcr7-/- embryos, compared to controls. Lung branching morphogenesis was comparable between Dhcr7-/- and controls at early stages, but delayed saccular development was visible in all Dhcr7-/- embryos from E17.5 onwards. Impaired pre-alveolar development of varying severity, inhibited cell proliferation, delayed differentiation of type I alveolar epithelial cells (AECs) and delayed vascular development were all evident in knockout lungs. Differentiation of type II AECs was apparently normal as judged by surfactant protein (SP) mRNAs and SP-C immunostaining. A significant amount of cholesterol was detectable in knockout lungs, implicating some maternal transfer of cholesterol. No significant differences of the spatial-temporal localization of sonic hedgehog (Shh) or its downstream targets by immunohistochemistry were detected between knockout and wild-type lungs and Shh autoprocessing occurred normally in tissues from Dhcr7-/- embryos. CONCLUSION: Our data indicated that cholesterol deficiency caused by Dhcr7 null was associated with a distinct lung saccular hypoplasia, characterized by failure to terminally differentiate alveolar sacs, a delayed differentiation of type I AECs and an immature vascular network at late gestational stages. The molecular mechanism of impaired lung development associated with sterol deficiency by Dhcr7 loss is still unknown, but these results do not support the involvement of dysregulated Shh-Patched-Gli pathway in causing this defect.

Origin of coronary endothelial cells from epicardial mesothelium in avian embryos.

It has been established that coronary vessels develop through self-assembly of mesenchymal vascular progenitors in the subepicardium. Mesenchymal precursors of vascular smooth muscle cells and fibroblasts are known to originate from an epithelial-to-mesenchymal transformation of the epicardial mesothelium, but the origin of the coronary endothelium is still obscure. We herein report that at least part of the population of the precursors of the coronary endothelium are epicardially-derived cells (EPDCs). We have performed an EPDC lineage study through retroviral and fluorescent labelling of the proepicardial and epicardial mesothelium of avian embryos. In all the experiments onlythe surface mesothelium was labelled after 3 h of reincubation. However, endothelial cells from subepicardial vessels were labelled after 24-48 h and endothelial cells of intramyocardial vessels were also labelled after 48-96 h of reincubation. In addition, the development of the coronary vessels was studied in quail-chick chimeras, obtaining results which also support a mesothelial origin for endothelial and smooth muscle cells. Finally, quail proepicardial explants cultured on Matrigel showed colocalization of cytokeratin and QH1 (mesothelial and endothelial markers, respectively) after 24 h. These results, taken together, suggest that EPDC show similar competence to that displayed by bipotential vascular progenitor cells [Yamashita et al., Nature 408: 92-96 (2000)] which are able to differentiate into endothelium or smooth muscle depending on their exposure to VEGF or PDGF-BB. It is conceivable that the earliest EPDC differentiate into endothelial cells in response to myocardially-secreted VEGF, while further EPDC would be recruited by the nascent capillaries via PDGFR-beta signalling, giving rise to mural cells.