Christina M. Alfieri

Regulation of Cardiomyocyte Proliferation and Myocardial Growth During Development by FOXO Transcription Factors

Cardiomyocytes actively proliferate during embryogenesis and withdraw from the cell cycle during neonatal stages. FOXO (Forkhead O) transcription factors are a direct target of phosphatidylinositol-3 kinase/AKT signaling in skeletal and smooth muscle and regulate expression of the Cip/Kip family of cyclin kinase inhibitors in other cell types; however, the interaction of phosphatidylinositol-3 kinase/AKT signaling, FOXO transcription factors, and cyclin kinase inhibitor expression has not been reported for the developing heart. Here, we show that FOXO1 and FOXO3 are expressed in the developing myocardium concomitant with increased cyclin kinase inhibitor expression from embryonic to neonatal stages. Cell culture studies show that embryonic cardiomyocytes are responsive to insulin-like growth factor 1 stimulation, which results in the induction of the phosphatidylinositol-3 kinase/AKT pathway, cytoplasmic localization of FOXO proteins, and increased myocyte proliferation. Likewise, adenoviral-mediated expression of AKT promotes cardiomyocyte proliferation and cytoplasmic localization of FOXO. In contrast, increased expression of FOXO1 negatively affects myocyte proliferation. In vivo myocyte-specific transgenic expression of FOXO1 during heart development causes embryonic lethality at embryonic day 10.5 because of severe myocardial defects that coincide with premature activation of p21cip1, p27kip1, and p57kip2 and decreased myocyte proliferation. Transgenic expression of dominant negative FOXO1 in cardiomyocytes does not obviously affect heart development at embryonic day 10.5, but results in abnormal morphology of the myocardium by embryonic day 18.5 along with decreased cyclin kinase inhibitor expression and increased myocyte proliferation. These data support FOXO transcription factors as negative regulators of cardiomyocyte proliferation and promoters of neonatal cell cycle withdrawal during heart development.

Wnt signaling in heart valve development and osteogenic gene induction.

Wnt signaling mediated by beta-catenin has been implicated in early endocardial cushion development, but its roles in later stages of heart valve maturation and homeostasis have not been identified. Multiple Wnt ligands and pathway genes are differentially expressed during heart valve development. At E12.5, Wnt2 is expressed in cushion mesenchyme, whereas Wnt4 and Wnt9b are predominant in overlying endothelial cells. At E17.5, both Wnt3a and Wnt7b are expressed in the remodeling atrioventricular (AV) and semilunar valves. In addition, the TOPGAL Wnt reporter transgene is active throughout the developing AV and semilunar valves at E16.5, with more localized expression in the stratified valve leaflets after birth. In chicken embryo aortic valves, genes characteristic of osteogenic cell lineages including periostin, osteonectin, and Id2 are expressed specifically in the collagen-rich fibrosa layer at E14. Treatment of E14 aortic valve interstitial cells (VICs) in culture with osteogenic media results in increased expression of multiple genes associated with bone formation. Treatment of VIC with Wnt3a leads to nuclear localization of beta-catenin and induction of periostin and matrix gla protein but does not induce genes associated with later stages of osteogenesis. Together, these studies provide evidence for Wnt signaling as a regulator of endocardial cushion maturation as well as valve leaflet stratification, homeostasis, and pathogenesis.

Mouse heart valve structure and function: echocardiographic and morphometric analyses from the fetus through the aged adult

The purpose of this study is to provide standard echocardiographic and morphometric data for normal mouse valve structure and function from late fetal to aged adult stages. Cross-sectional, two-dimensional and Doppler transthoracic echocardiography was performed in C57BL6 mice anesthetized with 1% to 2% isoflurane at embryonic day 18.5 (late fetal), 10 days (neonate), 1 mo (juvenile), 2 mo (young adult), 9 mo (old adult), and 16 mo (aged adult). Normal annulus dimensions indexed to age or weight, and selected flow velocities, were established by echocardiography. After echocardiographic imaging, hearts were harvested and histological and morphometric analyses were performed. Morphometric analysis demonstrated a progressive valve thinning and elongation during the fetal and juvenile stages that plateaued during adult stages (ANOVA, P < 0.01); however, there was increased thickening of the hinge of the aortic valve with advanced age, reminiscent of human aortic valve sclerosis. There was no age-related calcification. The results of this study provide comprehensive echocardiographic and morphometric data for normal mouse valve structure and function from late fetal to aged adult stages and should prove useful as a reference standard for future studies using mouse models of progressive valve disease.

Differential activation of valvulogenic, chondrogenic, and osteogenic pathways in mouse models of myxomatous and calcific aortic valve disease

Studies of human diseased aortic valves have demonstrated increased expression of genetic markers of valve progenitors and osteogenic differentiation associated with pathogenesis. Three potential mouse models of valve disease were examined for cellular pathology, morphology, and induction of valvulogenic, chondrogenic, and osteogenic markers. Osteogenesis imperfecta murine (Oim) mice, with a mutation in Col1a2, have distal leaflet thickening and increased proteoglycan composition characteristic of myxomatous valve disease. Periostin null mice also exhibit dysregulation of the ECM with thickening in the aortic midvalve region, but do not have an overall increase in valve leaflet surface area. Klotho null mice are a model for premature aging and exhibit calcific nodules in the aortic valve hinge-region, but do not exhibit leaflet thickening, ECM disorganization, or inflammation. Oim/oim mice have increased expression of valve progenitor markers Twist1, Col2a1, Mmp13, Sox9 and Hapln1, in addition to increased Col10a1 and Asporin expression, consistent with increased proteoglycan composition. Periostin null aortic valves exhibit relatively normal gene expression with slightly increased expression of Mmp13 and Hapln1. In contrast, Klotho null aortic valves have increased expression of Runx2, consistent with the calcified phenotype, in addition to increased expression of Sox9, Col10a1, and osteopontin. Together these studies demonstrate that oim/oim mice exhibit histological and molecular characteristics of myxomatous valve disease and Klotho null mice are a new model for calcific aortic valve disease.

Loss of β-Catenin Promotes Chondrogenic Differentiation of Aortic Valve Interstitial Cells

Objective— The Wnt/&bgr;-catenin signaling pathway has been implicated in human heart valve disease and is required for early heart valve formation in mouse and zebrafish. However, the specific functions of Wnt/&bgr;-catenin signaling activity in heart valve maturation and maintenance in adults have not been determined previously. Approach and Results— Here, we show that Wnt/&bgr;-catenin signaling inhibits Sox9 nuclear localization and proteoglycan expression in cultured chicken embryo aortic valves. Loss of &bgr;-catenin in vivo in mice, using Periostin(Postn)Cre–mediated tissue-restricted loss of &bgr;-catenin (Ctnnb1) in valvular interstitial cells, leads to the formation of aberrant chondrogenic nodules and induction of chondrogenic gene expression in adult aortic valves. These nodular cells strongly express nuclear Sox9 and Sox9 downstream chondrogenic extracellular matrix genes, including Aggrecan, Col2a1, and Col10a1. Excessive chondrogenic proteoglycan accumulation and disruption of stratified extracellular matrix maintenance in the aortic valve leaflets are characteristics of myxomatous valve disease. Both in vitro and in vivo data demonstrate that the loss of Wnt/&bgr;-catenin signaling leads to increased nuclear expression of Sox9 concomitant with induced expression of chondrogenic extracellular matrix proteins. Conclusions— &bgr;-Catenin limits Sox9 nuclear localization and inhibits chondrogenic differentiation during valve development and in adult aortic valve homeostasis.

Notch-Tnf signalling is required for development and homeostasis of arterial valves

Aims Congenital anomalies of arterial valves are common birth defects, leading to valvar stenosis. With no pharmaceutical treatment that can prevent the disease progression, prosthetic replacement is the only choice of treatment, incurring considerable morbidity and mortality. Animal models presenting localized anomalies and stenosis of congenital arterial valves similar to that of humans are critically needed research tools to uncover developmental molecular mechanisms underlying this devastating human condition. Methods and results We generated and characterized mouse models with conditionally altered Notch signalling in endothelial or interstitial cells of developing valves. Mice with inactivation of Notch1 signalling in valvar endothelial cells (VEC) developed congenital anomalies of arterial valves including bicuspid aortic valves and valvar stenosis. Notch1 signalling in VEC was required for repressing proliferation and activating apoptosis of valvar interstitial cells (VIC) after endocardial-to-mesenchymal transformation (EMT). We showed that Notch signalling regulated Tnf&agr; expression in vivo, and Tnf signalling was necessary for apoptosis of VIC and post-EMT development of arterial valves. Furthermore, activation or inhibition of Notch signalling in cultured pig aortic VEC-promoted or suppressed apoptosis of VIC, respectively. Conclusion We have now met the need of critical animal models and shown that Notch-Tnf signalling balances proliferation and apoptosis for post-EMT development of arterial valves. Our results suggest that mutations in its components may lead to congenital anomaly of aortic valves and valvar stenosis in humans.

Endothelial Cell Lineage Analysis Does Not Provide Evidence for EMT in Adult Valve Homeostasis and Disease: DOES EMT OCCUR IN ADULT HEART VALVES?

Epithelial‐to‐mesenchymal transition (EMT) enables stationary epithelial cells to exhibit migratory behavior and is the key step that initiates heart valve development. Recent studies suggest that EMT is reactivated in the pathogenesis of myxomatous valve disease (MVD), a condition that involves the progressive degeneration and thickening of valve leaflets. These studies have been limited to in vitro experimentation and reliance on histologic costaining of epithelial and mesenchymal markers as evidence of EMT in mouse and sheep models of valve disease. However, longitudinal analysis of cell lineage origins and potential pathogenic or reparative contributions of newly generated mesenchymal cells have not been reported previously. In this study, a genetic lineage tracing strategy was pursued by irreversibly labeling valve endothelial cells in the Osteogenesis imperfecta and Marfan syndrome mouse models to determine whether they undergo EMT during valve disease. Tie2‐CreER T2 and Cdh5(PAC)‐CreER T2 mouse lines were used in combination with colorimetric and fluorescent reporters for longitudinal assessment of endothelial cells. These lineage tracing experiments showed no evidence of EMT during adult valve homeostasis or valve pathogenesis. Additionally, CD31 and smooth muscle α‐actin (αSMA) double‐positive cells, used as an indicator of EMT, were not detected, and levels of EMT transcription factors were not altered. Interestingly, contrary to the endothelial cell‐specific Cdh5(PAC)‐CreER T2 driver line, Tie2‐CreER T2 lineage‐derived cells in diseased heart valves also included CD45+ leukocytes. Altogether, our data indicate that EMT is not a feature of valve homeostasis and disease but that increased immune cells may contribute to MVD. Anat Rec, 302:125–135, 2019.

Molecular Mechanisms of Heart Valve Development and Disease

The mature heart valves consist of stratified extracellular matrix (ECM) layers, and heart valve disease is characterized by ECM dysregulation and mineralization. There is increasing evidence that regulatory pathways that control heart valve development also are active in disease. In human diseased valves and mouse models, the expression of valve progenitor markers, including Twist1, Msx1/2 and Snail1/2, is induced. Additional markers of osteogenesis, including Runx2, osteocalcin and bone sialoprotein, also are expressed in calcific aortic valve disease (CAVD) in humans and mice. New mouse models have been developed for studies of valve disease mechanisms. Klotho-null mice are a model for premature aging and exhibit calcified nodules in aortic valves with osteogenic gene induction. Osteogenesis Imperfecta mice, bearing a collagen1a2 mutation, develop features of myxomatous valve disease, including thickening, increased proteoglycan deposition and chondrogenic gene induction. Together, these findings demonstrate specific molecular indicators of valve disease progression, including the identification of early disease markers, which represent potential targets for therapeutic intervention.