Wnt Signaling Inhibition Prevents Postnatal Inflammation and Disease Progression in Mouse Congenital Myxomatous Valve Disease.

BACKGROUND: Myxomatous valve disease (MVD) is the most common cause of mitral regurgitation, leading to impaired cardiac function and heart failure. MVD in a mouse model of Marfan syndrome includes valve leaflet thickening and progressive valve degeneration. However, the underlying mechanisms by which the disease progresses remain undefined. METHODS: Mice with Fibrillin 1 gene variant Fbn1C1039G/+ recapitulate histopathologic features of Marfan syndrome, and Wnt (Wingless-related integration site) signaling activity was detected in TCF/Lef-lacZ (T-cell factor/lymphoid enhancer factor-ß-galactosidase) reporter mice. Single-cell RNA sequencing was performed from mitral valves of wild-type and Fbn1C1039G/+ mice at 1 month of age. Inhibition of Wnt signaling was achieved by conditional induction of the secreted Wnt inhibitor Dkk1 (Dickkopf-1) expression in periostin-expressing valve interstitial cells of Periostin-Cre; tetO-Dkk1; R26rtTA; TCF/Lef-lacZ; Fbn1C1039G/+ mice. Dietary doxycycline was administered for 1 month beginning with MVD initiation (1-month-old) or MVD progression (2-month-old). Histological evaluation and immunofluorescence for ECM (extracellular matrix) and immune cells were performed. RESULTS: Wnt signaling is activated early in mitral valve disease progression, before immune cell infiltration in Fbn1C1039G/+ mice. Single-cell transcriptomics revealed similar mitral valve cell heterogeneity between wild-type and Fbn1C1039G/+ mice at 1 month of age. Wnt pathway genes were predominantly expressed in valve interstitial cells and valve endothelial cells of Fbn1C1039G/+ mice. Inhibition of Wnt signaling in Fbn1C1039G/+ mice at 1 month of age prevented the initiation of MVD as indicated by improved ECM remodeling and reduced valve leaflet thickness with decreased infiltrating macrophages. However, later, Wnt inhibition starting at 2 months did not prevent the progression of MVD. CONCLUSIONS: Wnt signaling is involved in the initiation of mitral valve abnormalities and inflammation but is not responsible for later-stage valve disease progression once it has been initiated. Thus, Wnt signaling contributes to MVD progression in a time-dependent manner and provides a promising therapeutic target for the early treatment of congenital MVD in Marfan syndrome.

Macrophage lineages in heart development and regeneration.

During development, macrophage subpopulations derived from hematopoietic progenitors take up residence in the developing heart. Embryonic macrophages are detectable at the early stages of heart formation in the nascent myocardium, valves and coronary vasculature. The specific subtypes of macrophages present in the developing heart reflect the generation of hematopoietic progenitors in the yolk sac, aorta-gonad-mesonephros, fetal liver, and postnatal bone marrow. Ablation studies have demonstrated specific requirements for embryonic macrophages in valve remodeling, coronary and lymphatic vessel development, specialized conduction system maturation, and myocardial regeneration after neonatal injury. The developmental origins of macrophage lineages change over time, with embryonic lineages having more reparative and remodeling functions in comparison to the bone marrow derived myeloid lineages of adults. Here we review the contributions and functions of cardiac macrophages in the developing heart with potential regenerative and reparative implications for cardiovascular disease.

Dynamic changes in mitral valve extracellular matrix, tissue mechanics and function in a mouse model of Marfan syndrome.

OBJECTIVE: Mouse models of Marfan syndrome (MFS) with Fibrillin 1 (Fbn1) variant C1041G exhibit cardiovascular abnormalities, including myxomatous valve disease (MVD) and aortic aneurism, with structural extracellular matrix (ECM) dysregulation. In this study, we examine the structure-function-mechanics relations of the mitral valve related to specific transitions in ECM composition and organization in progressive MVD in MFS mice from Postnatal day (P)7 to 1 year-of-age. APPROACH AND RESULTS: Mechanistic links between mechanical forces and biological changes in MVD progression were examined in Fbn1C1041G/+ MFS mice. By echocardiography, mitral valve dysfunction is prevalent at 2 months with a decrease in cardiac function at 6 months, followed by a preserved cardiac function at 12 months. Mitral valve (MV) regurgitation occurs in a subset of mice at 2-6 months, while progressive dilatation of the aorta occurs from 2 to 12 months. Mitral valve tissue mechanical assessments using a uniaxial Permeabilizable Fiber System demonstrate decreased stiffness of MFS MVs at all stages. Histological and microscopic analysis of ECM content, structure, and fiber orientation demonstrate that alterations in ECM mechanics, composition, and organization precede functional abnormalities in Fbn1C1041G/+MFS MVs. At 2 months, ECM abnormalities are detected with an increase in proteoglycans and decreased stiffness of the mitral valve. By 6-12 months, collagen fiber remodeling is increased with abnormal fiber organization in MFS mitral valve leaflets. At the same time, matrifibrocyte gene expression characteristic of collagen-rich connective tissue is increased, as detected by RNA in situ hybridization and qPCR. Together, these studies demonstrate early prevalence of proteoglycans at 2 months followed by upregulation of collagen structure and organization with age in MVs of MFS mice. CONCLUSIONS: Altogether, our data indicate dynamic regulation of mitral valve structure, tissue mechanics, and function that reflect changes in ECM composition, organization, and gene expression in progressive MVD. Notably, increased collagen fiber organization and orientation, potentially dependent on increased matrifibrocyte cell activity, is apparent with altered mitral valve mechanics and function in aging MFS mice.

PROX1 Inhibits PDGF-B Expression to Prevent Myxomatous Degeneration of Heart Valves.

BACKGROUND: Cardiac valve disease is observed in 2.5% of the general population and 10% of the elderly people. Effective pharmacological treatments are currently not available, and patients with severe cardiac valve disease require surgery. PROX1 (prospero-related homeobox transcription factor 1) and FOXC2 (Forkhead box C2 transcription factor) are transcription factors that are required for the development of lymphatic and venous valves. We found that PROX1 and FOXC2 are expressed in a subset of valvular endothelial cells (VECs) that are located on the downstream (fibrosa) side of cardiac valves. Whether PROX1 and FOXC2 regulate cardiac valve development and disease is not known. METHODS: We used histology, electron microscopy, and echocardiography to investigate the structure and functioning of heart valves from Prox1ΔVEC mice in which Prox1 was conditionally deleted from VECs. Isolated valve endothelial cells and valve interstitial cells were used to identify the molecular mechanisms in vitro, which were tested in vivo by RNAScope, additional mouse models, and pharmacological approaches. The significance of our findings was tested by evaluation of human samples of mitral valve prolapse and aortic valve insufficiency. RESULTS: Histological analysis revealed that the aortic and mitral valves of Prox1ΔVEC mice become progressively thick and myxomatous. Echocardiography revealed that the aortic valves of Prox1ΔVEC mice are stenotic. FOXC2 was downregulated and PDGF-B (platelet-derived growth factor-B) was upregulated in the VECs of Prox1ΔVEC mice. Conditional knockdown of FOXC2 and conditional overexpression of PDGF-B in VECs recapitulated the phenotype of Prox1ΔVEC mice. PDGF-B was also increased in mice lacking FOXC2 and in human mitral valve prolapse and insufficient aortic valve samples. Pharmacological inhibition of PDGF-B signaling with imatinib partially ameliorated the valve defects of Prox1ΔVEC mice. CONCLUSIONS: PROX1 antagonizes PDGF-B signaling partially via FOXC2 to maintain the extracellular matrix composition and prevent myxomatous degeneration of cardiac valves.

Localized Prox1 Regulates Aortic Valve Endothelial Cell Diversity and Extracellular Matrix Stratification in Mice.

BACKGROUND: Specialized valve endothelial cell (VEC) populations are localized oriented to blood flow in developing aortic and mitral valves, but their roles in valve development and disease are unknown. In the aortic valve (AoV), a population of VECs on the fibrosa side expresses the transcription factor Prox1 together with genes found in lymphatic ECs. In this study, we examine Prox1's role in regulating a lymphatic-like gene network and promoting VEC diversity required for the development of the stratified trilaminar extracellular matrix (ECM) of murine AoV leaflets. METHODS: To determine whether disruption of Prox1 localization affects heart valve development, we generated mice (NFATc1enCre Prox1 gain-of-function) in which Prox1 is overexpressed on the ventricularis side of the AoV beginning in embryonic development. To identify potential targets of Prox1, we performed cleavage under targets and release using nuclease on wild-type and NFATc1enCre Prox1 gain-of-function AoVs with validation by colocalization in vivo using RNA in situ hybridization in NFATc1enCre Prox1 gain-of-function AoVs. Natural induction of Prox1 and target gene expression was evaluated in myxomatous AoVs in a mouse model of Marfan syndrome (Fbn1C1039G/+). RESULTS: The overexpression of Prox1 is sufficient to cause enlargement of AoVs by postnatal day (P)0, as well as a decrease in ventricularis-specific gene expression and disorganized interstitial ECM layers at P7. We identified potential targets of Prox1 known to play roles in lymphatic ECs including Flt1, Efnb2, Egfl7, and Cx37. Ectopic Prox1 colocalized with induced Flt1, Efnb2, and Cx37 expression in NFATc1enCre Prox1 gain-of-function AoVs. Moreover, in Marfan syndrome myxomatous AoVs, endogenous Prox1, and its identified targets, were ectopically induced in ventricularis side VECs. CONCLUSIONS: Our results support a role for Prox1 in localized lymphatic-like gene expression on the fibrosa side of the AoV. Furthermore, localized VEC specialization is required for development of the stratified trilaminar ECM critical for AoV function and is dysregulated in congenitally malformed valves.

Btg1 and Btg2 regulate neonatal cardiomyocyte cell cycle arrest.

Rodent cardiomyocytes undergo mitotic arrest in the first postnatal week. Here, we investigate the role of transcriptional co-regulator Btg2 (B-cell translocation gene 2) and functionally-similar homolog Btg1 in postnatal cardiomyocyte cell cycling and maturation. Btg1 and Btg2 (Btg1/2) are expressed in neonatal C57BL/6 mouse left ventricles coincident with cardiomyocyte cell cycle arrest. Btg1/2 constitutive double knockout (DKO) mouse hearts exhibit increased pHH3+ mitotic cardiomyocytes compared to Wildtype at postnatal day (P)7, but not at P30. Similarly, neonatal AAV9-mediated Btg1/2 double knockdown (DKD) mouse hearts exhibit increased EdU+ mitotic cardiomyocytes compared to Scramble AAV9-shRNA controls at P7, but not at P14. In neonatal rat ventricular myocyte (NRVM) cultures, siRNA-mediated Btg1/2 single and double knockdown cohorts showed increased EdU+ cardiomyocytes compared to Scramble siRNA controls, without increase in binucleation or nuclear DNA content. RNAseq analyses of Btg1/2-depleted NRVMs support a role for Btg1/2 in inhibiting cell proliferation, and in modulating reactive oxygen species response pathways, implicated in neonatal cardiomyocyte cell cycle arrest. Together, these data identify Btg1 and Btg2 as novel contributing factors in mammalian cardiomyocyte cell cycle arrest after birth.

Therapeutic CCR2 Blockade Prevents Inflammation and Alleviates Myxomatous Valve Disease in Marfan Syndrome.

Myxomatous valve disease (MVD) can lead to cardiac dysfunction and heart failure, yet medical therapies are lacking. C-C chemokine receptor type 2 (CCR2)+ immune cell infiltration promotes mitral valve inflammation in a Marfan syndrome (MFS) mouse model. The CCR2 genetic knockout reduces inflammation with downregulated proteases and improved extracellular matrix integrity. Pharmacological inhibition of CCR2+ cell infiltration by RS504393 prevents the initiation and progression of MVD, indicated by restored protease expression, improved extracellular matrix organization, and reduced valve leaflet thickness in MFS mice. Thus, the CCR2 antagonist RS504393 is a promising therapy for the treatment of MVD in MFS.

Transcriptional Regulation of Postnatal Cardiomyocyte Maturation and Regeneration.

During the postnatal period, mammalian cardiomyocytes undergo numerous maturational changes associated with increased cardiac function and output, including hypertrophic growth, cell cycle exit, sarcomeric protein isoform switching, and mitochondrial maturation. These changes come at the expense of loss of regenerative capacity of the heart, contributing to heart failure after cardiac injury in adults. While most studies focus on the transcriptional regulation of embryonic or adult cardiomyocytes, the transcriptional changes that occur during the postnatal period are relatively unknown. In this review, we focus on the transcriptional regulators responsible for these aspects of cardiomyocyte maturation during the postnatal period in mammals. By specifically highlighting this transitional period, we draw attention to critical processes in cardiomyocyte maturation with potential therapeutic implications in cardiovascular disease.

Periostin-expressing Schwann cells and endoneurial cardiac fibroblasts contribute to sympathetic nerve fasciculation after birth.

BACKGROUND: The intracardiac nervous system (ICNS) is composed of neurons, in association with Schwann cells (SC) and endoneurial cardiac fibroblasts (ECF). Besides heart rhythm control, recent studies have implicated cardiac nerves in postnatal cardiac regeneration and cardiomyocyte size regulation, but cardiac SC and ECF remain understudied. During the postnatal period, the ICNS undergoes intense remodeling with nerve fasciculation and elongation throughout the myocardium, partially guided by the extracellular matrix (ECM). Here we report the origins, heterogeneity, and functions of SC and ECF that develop in proximity to neurons during postnatal ICNS maturation. METHODS AND RESULTS: Periostin lineage (Postn+) cells include cardiac Remak SC and ECF during the postnatal period in mice. The developmental origins of cardiac SC and ECF were examined using Rosa26eGFP reporter mice bred with Wnt1Cre, expressed in Neural crest (NC)-derived lineages, or tamoxifen-inducible Tcf21MerCreMer, expressed predominantly in epicardial-derived fibroblast lineages. ICNS components are NC-derived with the exceptions of the myelinating Plp1+ SC and the Tcf21+ lineage-derived intramural ventricular ECF. In addition, Postn+ lineage GFAP- Remak SC and ECF are present around the fasciculating cardiac nerves. Whole mount studies of the NC-derived cells confirmed postnatal maturation of the complex ICNS network from P0 to P30. Sympathetic, parasympathetic, and sensory neurons fasciculate from P0 to P7 indicated by co-staining with PSA-NCAM. Ablation of Postn+ cells from P0 to P6 or loss of Periostin leads to reduced fasciculation of cardiac sympathetic nerves. In addition, collagen remodeling surrounding maturing nerves of the postnatal heart is reduced in Postn-null mice. CONCLUSIONS: Postn+ cells include cardiac SC and ECF during postnatal nerve maturation, and these cells have different embryonic origins. At P7, the Postn+ cells associated with cardiac nerves are mainly Remak SC and ECF. Ablation of the Postn+ cells from P0 to P6 and also loss of Postn in Postn-null mice leads to reduced fasciculation of cardiac nerves at P7.

Macrophage lineages in heart valve development and disease.

Heterogeneous macrophage lineages are present in the aortic and mitral valves of the heart during development and disease. These populations include resident macrophages of embryonic origins and recruited monocyte-derived macrophages prevalent in disease. Soon after birth, macrophages from haematopoietic lineages are recruited to the heart valves, and bone marrow transplantation studies in mice demonstrate that haematopoietic-derived macrophages continue to invest adult valves. During myxomatous heart valve disease, monocyte-derived macrophages are recruited to the heart valves and they contribute to valve degeneration in a mouse model of Marfan syndrome. Here, we review recent studies of macrophage lineages in heart valve development and disease with discussion of clinical significance and therapeutic applications.

Ube2v1 Positively Regulates Protein Aggregation by Modulating Ubiquitin Proteasome System Performance Partially Through K63 Ubiquitination.

RATIONALE: Compromised protein quality control can result in proteotoxic intracellular protein aggregates in the heart, leading to cardiac disease and heart failure. Defining the participants and understanding the underlying mechanisms of cardiac protein aggregation is critical for seeking therapeutic targets. We identified Ube2v1 (ubiquitin-conjugating enzyme E2 variant 1) in a genome-wide screen designed to identify novel effectors of the aggregation process. However, its role in the cardiomyocyte is undefined. OBJECTIVE: To assess whether Ube2v1 regulates the protein aggregation caused by cardiomyocyte expression of a mutant αB crystallin (CryABR120G) and identify how Ube2v1 exerts its effect. METHODS AND RESULTS: Neonatal rat ventricular cardiomyocytes were infected with adenoviruses expressing either wild-type CryAB (CryABWT) or CryABR120G. Subsequently, loss- and gain-of-function experiments were performed. Ube2v1 knockdown decreased aggregate accumulation caused by CryABR120G expression. Overexpressing Ube2v1 promoted aggregate formation in CryABWT and CryABR120G-expressing neonatal rat ventricular cardiomyocytes. Ubiquitin proteasome system performance was analyzed using a ubiquitin proteasome system reporter protein. Ube2v1 knockdown improved ubiquitin proteasome system performance and promoted the degradation of insoluble ubiquitinated proteins in CryABR120G cardiomyocytes but did not alter autophagic flux. Lys (K) 63-linked ubiquitination modulated by Ube2v1 expression enhanced protein aggregation and contributed to Ube2v1's function in regulating protein aggregate formation. Knocking out Ube2v1 exclusively in cardiomyocytes by using AAV9 (adeno-associated virus 9) to deliver multiplexed single guide RNAs against Ube2v1 in cardiac-specific Cas9 mice alleviated CryABR120G-induced protein aggregation, improved cardiac function, and prolonged lifespan in vivo. CONCLUSIONS: Ube2v1 plays an important role in protein aggregate formation, partially by enhancing K63 ubiquitination during a proteotoxic stimulus. Inhibition of Ube2v1 decreases CryABR120G-induced aggregate formation through enhanced ubiquitin proteasome system performance rather than autophagy and may provide a novel therapeutic target to treat cardiac proteinopathies.

Deficiency of Circulating Monocytes Ameliorates the Progression of Myxomatous Valve Degeneration in Marfan Syndrome.

BACKGROUND: Myxomatous valve degeneration (MVD) involves the progressive thickening and degeneration of the heart valves, leading to valve prolapse, regurgitant blood flow, and impaired cardiac function. Leukocytes composed primarily of macrophages have recently been detected in myxomatous valves, but the timing of the presence and the contributions of these cells in MVD progression are not known. METHODS: We examined MVD progression, macrophages, and the valve microenvironment in the context of Marfan syndrome (MFS) using mitral valves from MFS mice (Fbn1C1039G/+), gene-edited MFS pigs (FBN1Glu433AsnfsX98/+), and patients with MFS. Additional histological and transcriptomic evaluation was performed by using nonsyndromic human and canine myxomatous valves, respectively. Macrophage ontogeny was determined using MFS mice transplanted with mTomato+ bone marrow or MFS mice harboring RFP (red fluorescent protein)-tagged C-C chemokine receptor type 2 (CCR2) monocytes. Mice deficient in recruited macrophages (Fbn1C1039G/+;Ccr2RFP/RFP) were generated to determine the requirements of recruited macrophages to MVD progression. RESULTS: MFS mice recapitulated histopathological features of myxomatous valve disease by 2 months of age, including mitral valve thickening, increased leaflet cellularity, and extracellular matrix abnormalities characterized by proteoglycan accumulation and collagen fragmentation. Diseased mitral valves of MFS mice concurrently exhibited a marked increase of infiltrating (MHCII+, CCR2+) and resident macrophages (CD206+, CCR2-), along with increased chemokine activity and inflammatory extracellular matrix modification. Likewise, mitral valve specimens obtained from gene-edited MFS pigs and human patients with MFS exhibited increased monocytes and macrophages (CD14+, CD64+, CD68+, CD163+) detected by immunofluorescence. In addition, comparative transcriptomic evaluation of both genetic (MFS mice) and acquired forms of MVD (humans and dogs) unveiled a shared upregulated inflammatory response in diseased valves. Remarkably, the deficiency of monocytes was protective against MVD progression, resulting in a significant reduction of MHCII macrophages, minimal leaflet thickening, and preserved mitral valve integrity. CONCLUSIONS: All together, our results suggest sterile inflammation as a novel paradigm to disease progression, and we identify, for the first time, monocytes as a viable candidate for targeted therapy in MVD.

Calcification and extracellular matrix dysregulation in human postmortem and surgical aortic valves.

OBJECTIVES: Calcific aortic valve disease (CAVD) is a progressive disease ranging from aortic valve (AoV) sclerosis to AoV stenosis (AS), characterised by severe calcification with impaired leaflet function. Due to the lack of early symptoms, the pathological progression towards valve dysfunction is poorly understood. The early patterns of AoV calcification and altered extracellular matrix (ECM) organisation were analysed in individuals postmortem without clinical AS compared with clinical AS. METHODS: Histological patterns of calcification and ECM organisation in postmortem AoV leaflets without clinical AS obtained from a tissue repository and surgical specimens obtained from individuals with clinical AS were compared with in vivo imaging prior to transcatheter AoV implantation. RESULTS: AoV calcification was detected in all samples from individuals >50 years old, with severity increasing with age, independent of known CAVD risk factors. Two distinct types of calcification were identified: 'Intrinsic', primarily found at the leaflet hinge of postmortem leaflets, accompanied by abnormal collagen and proteoglycan deposition; and 'Nodular', extending from the middle to the tip regions in more severely affected postmortem leaflets and surgical specimens, associated with increased elastin fragmentation and loss of elastin integrity. Even in the absence of increased thickening, abnormalities in ECM composition were observed in postmortem leaflets without clinical AS and worsen in clinical AS. CONCLUSIONS: Two distinct phenotypes of AoV calcification are apparent. While the 'nodular' form is recognised on in vivo imaging and is present with CAVD and valve dysfunction, it is unclear if the 'intrinsic' form is pathological or detected on in vivo imaging.

Endothelial Cell Lineage Analysis Does Not Provide Evidence for EMT in Adult Valve Homeostasis and Disease.

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. © 2018 Wiley Periodicals, Inc.

Macrophage Transitions in Heart Valve Development and Myxomatous Valve Disease.

OBJECTIVE: Hematopoietic-derived cells have been reported in heart valves but remain poorly characterized. Interestingly, recent studies reveal infiltration of leukocytes and increased macrophages in human myxomatous mitral valves. Nevertheless, timing and contribution of macrophages in normal valves and myxomatous valve disease are still unknown. The objective is to characterize leukocytes during postnatal heart valve maturation and identify macrophage subsets in myxomatous valve disease. APPROACH AND RESULTS: Leukocytes are detected in heart valves after birth, and their numbers increase during postnatal valve development. Flow cytometry and immunostaining analysis indicate that almost all valve leukocytes are myeloid cells, consisting of at least 2 differentially localized macrophage subsets and dendritic cells. Beginning a week after birth, increased numbers of CCR2+ (C-C chemokine receptor type 2) macrophages are present, consistent with infiltrating populations of monocytes, and macrophages are localized in regions of biomechanical stress in the valve leaflets. Valve leukocytes maintain expression of CD (cluster of differentiation) 45 and do not contribute to significant numbers of endothelial or interstitial cells. Macrophage lineages were examined in aortic and mitral valves of Axin2 KO (knockout) mice that exhibit myxomatous features. Infiltrating CCR2+ monocytes and expansion of CD206-expressing macrophages are localized in regions where modified heavy chain hyaluronan is observed in myxomatous valve leaflets. Similar colocalization of modified hyaluronan and increased numbers of macrophages were observed in human myxomatous valve disease. CONCLUSIONS: Our study demonstrates the heterogeneity of myeloid cells in heart valves and highlights an alteration of macrophage subpopulations, notably an increased presence of infiltrating CCR2+ monocytes and CD206+ macrophages, in myxomatous valve disease.

Hypoxia promotes primitive glycosaminoglycan-rich extracellular matrix composition in developing heart valves.

During postnatal heart valve development, glycosaminoglycan (GAG)-rich valve primordia transform into stratified valve leaflets composed of GAGs, fibrillar collagen, and elastin layers accompanied by decreased cell proliferation as well as thinning and elongation. The neonatal period is characterized by the transition from a uterine environment to atmospheric O2, but the role of changing O2 levels in valve extracellular matrix (ECM) composition or morphogenesis is not well characterized. Here, we show that tissue hypoxia decreases in mouse aortic valves in the days after birth, concomitant with ECM remodeling and cell cycle arrest of valve interstitial cells. The effects of hypoxia on late embryonic valve ECM composition, Sox9 expression, and cell proliferation were examined in chicken embryo aortic valve organ cultures. Maintenance of late embryonic chicken aortic valve organ cultures in a hypoxic environment promotes GAG expression, Sox9 nuclear localization, and indicators of hyaluronan remodeling but does not affect fibrillar collagen content or cell proliferation. Chronic hypoxia also promotes GAG accumulation in murine adult heart valves in vivo. Together, these results support a role for hypoxia in maintaining a primitive GAG-rich matrix in developing heart valves before birth and also in the induction of hyaluronan remodeling in adults.NEW & NOTEWORTHY Tissue hypoxia decreases in mouse aortic valves after birth, and exposure to hypoxia promotes glycosaminoglycan accumulation in cultured chicken embryo valves and adult murine heart valves. Thus, hypoxia maintains a primitive extracellular matrix during heart valve development and promotes extracellular matrix remodeling in adult mice, as occurs in myxomatous disease.

Loss of Axin2 results in impaired heart valve maturation and subsequent myxomatous valve disease.

AIMS: Myxomatous valve disease (MVD) is the most common aetiology of primary mitral regurgitation. Recent studies suggest that defects in heart valve development can lead to heart valve disease in adults. Wnt/ß-catenin signalling is active during heart valve development and has been reported in human MVD. The consequences of increased Wnt/ß-catenin signalling due to Axin2 deficiency in postnatal valve remodelling and pathogenesis of MVD were determined. METHODS AND RESULTS: To investigate the role of Wnt/ß-catenin signalling, we analysed heart valves from mice deficient in Axin2 (KO), a negative regulator of Wnt/ß-catenin signalling. Axin2 KO mice display enlarged mitral and aortic valves (AoV) after birth with increased Wnt/ß-catenin signalling and cell proliferation, whereas Sox9 expression and collagen deposition are decreased. At 2 months in Axin2 KO mice, the valve extracellular matrix (ECM) is stratified but distal AoV leaflets remain thickened and develop aortic insufficiency. Progressive myxomatous degeneration is apparent at 4 months with extensive ECM remodelling and focal aggrecan-rich areas, along with increased BMP signalling. Infiltration of inflammatory cells is also observed in Axin2 KO AoV prior to ECM remodelling. Overall, these features are consistent with the progression of human MVD. Finally, Axin2 expression is decreased and Wnt/ß-catenin signalling is increased in myxomatous mitral valves in a murine model of Marfan syndrome, supporting the importance of Wnt/ß-catenin signalling in the development of MVD. CONCLUSIONS: Altogether, these data indicate that Axin2 limits Wnt/ß-catenin signalling after birth and allows proper heart valve maturation. Moreover, dysregulation of Wnt/ß-catenin signalling resulting from loss of Axin2 leads to progressive MVD.

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 Tnfα 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.

Bone Morphogenetic Protein Signaling Is Required for Aortic Valve Calcification.

OBJECTIVE: Calcific aortic valve disease (CAVD) is the most prevalent type of heart valve disease, affecting ≈2% of the US population. CAVD is characterized by the presence of calcific nodules, resulting in aortic valve (AoV) stenosis; however, the underlying mechanisms driving disease remain unknown. Studies of human diseased AoV provide initial evidence that bone morphogenetic protein (BMP) signaling, essential for normal bone formation, is activated during CAVD. Mice deficient in Klotho, an FGF23 transmembrane coreceptor, exhibit premature aging and develop AoV calcific nodules as occurs in human CAVD. The role of BMP signaling in the development of CAVD was examined in porcine aortic valve interstitial cells (VICs) and Klotho(-/-) mice. APPROACH AND RESULTS: We show that activation of BMP signaling, as indicated by pSmad1/5/8 expression, precedes and later localizes with AoV calcification in Klotho(-/-) mice. In addition, cellular and extracellular matrix changes resembling features of normal bone formation are accompanied by increased osteochondrogenic gene induction in calcified Klotho(-/-) AoV. Likewise, osteogenic media treatment of porcine VICs results in BMP pathway activation, increased osteochondrogenic gene induction, and formation of calcific nodules in vitro. We demonstrate that genetic inactivation of the BMP type IA receptor in Klotho(-/-) aortic VICs, as well as BMP pathway inhibition of osteogenic media-treated aortic VICs in vitro, results in the inhibition of AoV calcification. CONCLUSIONS: BMP signaling and osteochondrogenic gene induction are active in calcified Klotho(-/-) AoV in vivo and calcified porcine aortic VICs in vitro. Importantly, BMP signaling is required for the development of AoV calcification in vitro and in vivo.

Overexpression of Tbx20 in Adult Cardiomyocytes Promotes Proliferation and Improves Cardiac Function After Myocardial Infarction.

BACKGROUND: Adult mammalian cardiomyocytes (CMs) have the potential to proliferate, but this is not sufficient to generate adequate CMs after myocardial infarction (MI). The transcription factor Tbx20 is required for CM proliferation during development and adult CM homeostasis. The ability of Tbx20 overexpression (Tbx20(OE)) to promote adult CM proliferation and to improve cardiac function after MI was examined. METHODS AND RESULTS: Tbx20(OE) was induced specifically in adult mouse differentiated CMs. Increased CM proliferation and fetal-like characteristics were found in Tbx20(OE) hearts compared with controls without causing pathology 4 weeks after Tbx20(OE) at baseline. Moreover, Tbx20(OE) in adult CM after MI significantly improved survival, cardiac function, and infarct size 4 weeks after MI. Improved cardiac repair, as indicated by increased CM proliferation and capillary density, was observed in the MI border zone of Tbx20(OE) hearts compared with controls. Expression of proliferation activator (cyclin D1, E1, and IGF1) and fetal contractile protein (ssTNI, ßMHC) mRNA was increased whereas negative cell-cycle regulators (p21, Meis1) were decreased in Tbx20(OE) hearts compared with controls under both baseline and MI conditions. Tbx20(OE) in adult hearts activates multiple proproliferation pathways, including Akt, YAP and BMP. Interestingly, p21, Meis1, and a novel cell-cycle inhibitory gene, Btg2, are directly bound and repressed by Tbx20 with induction of proliferation in neonatal CM. CONCLUSIONS: Tbx20(OE), specifically in adult CM, activates multiple cardiac proliferative pathways, directly represses cell-cycle inhibitory genes p21, Meis1, and Btg2, promotes adult CM proliferation; and preserves cardiac performance after MI.