You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: VGLL4 plays a critical role in heart valve development and homeostasis}
Article:
Abstract:
Heart valve disease is a major clinical problem worldwide. Cardiac valve development and homeostasis need to be precisely controlled. Hippo signaling is essential for organ development and tissue homeostasis, while its role in valve formation and morphology maintenance remains unknown. VGLL4 is a transcription cofactor in vertebrates and we found it was mainly expressed in valve interstitial cells at the post-EMT stage and was maintained till the adult stage. Tissue specific knockout of VGLL4 in different cell lineages revealed that only loss of VGLL4 in endothelial cell lineage led to valve malformation with expanded expression of YAP targets. We further semi-knockout YAP in VGLL4 ablated hearts, and found hyper proliferation of arterial valve interstitial cells was significantly constrained. These findings suggest that VGLL4 is important for valve development and manipulation of Hippo components would be a potential therapy for preventing the progression of congenital valve disease.
Introduction:
Heart valves are critical for orchestrating the unidirectional blood flow during the cardiac cycle, ensuring the correct and efficient function of the heart[1]. Heart valve disease has a high morbidity and mortality worldwide and their incidence increases with age[2]. Heart arterial valve development starts with endothelial-to-mesenchymal transition( EMT) to form the endocardial cushion, in which valvular endothelial cells( VEC) invade into the outflow tract( OFT) and trans-differentiate into valvar interstitial cells( VIC) to form the proximal cushions. Subsequently, neural crest cells migrate into the OFT to form the distal cushion. Then the valve primordia undergo extracellular matrix( ECM) remodeling and elongate into thin mature leaflets with three separate layers: the ventricularis( elastin-rich), fibrosa( fibrillar collagen-rich) and spongiosa( proteoglycan-rich). During this process, cell proliferation and apoptosis of VEC and VIC are precisely regulated, and, once disturbed, it will result in valve malformation[3, 4]. Valve disease can occur due to impaired cardiac cushion development, following infections or other elevated stress conditions such as heart attack, and usually lead to calcification or stenosis, eventually resulting in congestive heart failure[5]. However, the molecular mechanism underlying valve maturation and homeostasis remains incompletely understood so far. The Hippo pathway is an evolutionarily conserved pathway from Drosophila to human which regulates cell proliferation, differentiation, stem cell fate determination and organ size in multiple organs and tissues[6]. Numerous studies demonstrate that the Hippo pathway is not only critical for embryonic cardiac development, perinatal cardiomyocyte proliferation and heart regeneration[7–9], but also plays important roles in multiple cardiovascular diseases, such as myocardial infarction, cardiac hypertrophy and atherosclerosis[10–12]. The Vestigial-like( VGLL) family includes its four members VGLL1-4, which mainly serve as transcription cofactors in vertebrates[13]. We have previously identified VGLL4 in mice and its homolog SdBP in Drosophila that competes with YAP for TEADs binding, which results in the inhibition of YAP-induced overgrowth and tumor genesis in gastric and lung cancer[14–16]. Interestingly, VGLL4 is the only member of the VGLL family expressed in the heart[13]. While the role of VGLL4 in heart valve development and homeostasis has remained unknown. Here we constructed a VGLL4 reporter mouse line to investigate the VGLL4 expression pattern and found that the expression of VGLL4 increased dramatically in endothelial and interstitial cells in the valve region at the post-EMT stage and was maintained till the adult stage. We further used endothelial specific and neural crest specific Cre mice to knock out VGLL4 in different cell lineages that have been reported to contribute to valve interstitial cells. We found that only endothelial loss of VGLL4 led to valve malformation. Mechanistically, after semi-knockout of YAP, the phenotype of arterial valves in VGLL4 deleted heart was rescued, indicating that VGLL4 was required for normal valve development by antagonizing YAP activity. Together, we identified a key role of VGLL4 in valve development and maintenance.
Discussion:
In this study, we used mouse genetics to determine the function of VGLL4 in mouse heart valve development. The results revealed an essential role of VGLL4 in heart valve morphogenesis via regulating VIC proliferation and apoptosis. In particular, we showed that deletion of VGLL4 resulted in progressive valve disease. Even as early as E15. 5, Vgll4-/- AoV and PV exhibited dramatically VIC over-proliferation. Further studies showed that loss of VGLL4 in endothelial cells resulted in abnormal arterial valves morphology and developed severe cardiac hypertrophy in 36-week-old( aged) mice, while semi-knockout of YAP rescued heart malformation induced by VGLL4 deletion. Altogether, our study shows that VGLL4 and precisely regulated Hippo signaling are required for proper heart valve remodeling and maturation. The processes of valve development, remodeling and homeostasis are accompanied with cell proliferation, differentiation and apoptosis, which are precisely regulated by complex integration of multiple signaling pathways, such as Notch[20], Wingless-type MMTV integration site family( WNT)[21], Transforming Growth Factor-beta( TGFβ)[22], Bone Morphogenetic Protein( BMP)[23] and fibroblast growth factor( FGF)[24], vascular endothelial growth factor( VEGF)[25] pathways. The Hippo pathway plays important roles in cell differentiation, proliferation and apoptosis. YAP is a downstream effector of the Hippo pathway. However, the role of Hippo signaling in valve remodeling and morphology remains undisclosed. Previously, we reported that YAP was required for EMT during heart cushion formation at E9. 5. YAP knockout in endothelial cells reduced endocardial cell proliferation, led to hypocellular cardiac cushion and increased embryonic lethality[26]. In this study, we detected YAP remain widely expressed in post-EMT valves at E15. 5, enlighten us that YAP also plays a role in post EMT valve development. VGLL4, a transcriptional co-repressor in the Hippo pathway, is critical for balancing cell proliferation and apoptosis during development and tissue homeostasis. As VGLL4 and YAP don’t contain DNA-binding domain and they mediate their biological functions mainly through interaction with TEAD transcription factor family[27]. Our previous studies have shown that VGLL4 could bind with TEAD4 through its TDU domains and directly competed with YAP for binding to TEAD4, which result in significantly suppressing cell growth in gastric and lung cancer cell lines with decreased expression of YAP/TEAD downstream genes including CTGF and CYR61, whereas deletion of both TDU domains attenuated the inhibitory function[14–16]. So we think during valve development, VGLL4 also exerts its inhibitory function through the competition for YAP-TEAD complex formation, as endothelial knockout of VGLL4 leads to endothelial origin VIC over-proliferation, further bolsters the inverse correlation between VGLL4 and proliferation capacity. Based on our Vgll4-GFP reporter mice line we found that VGLL4 is extensively expressed in endothelial cells and valve region at E13. 5, which enlightens us that VGLL4 plays its role in the post-EMT stage in which proliferation needs to be restricted. At the same time, apoptosis starts and valves begin to remodel and elongate into a thin valve. At E15. 5, VGLL4 is expressed in all heart valves( AoV, PV, MV and TV), but restricted in AoV and PV and seldom detected in MV and TV at E17. 5( S10 Fig), this could explain our VGLL4 total knockout phenotype that MV and TV were normal on neonatal heart( S3 Fig). Moreover, from P0 to adult stage, VGLL4 is more inclined to locate in the fibrosa layer, the side in which the valve bears disturbed flow condition, where it is more likely for pathophysiologic changes such as sclerosis and calcification to occur, and where valve dysfunction, such as regurgitation and stenosis take place. The relationship between VGLL4 and fibrosa signaling in valve disease need to be further studied. From our lineage trace data, we disclosed that VGLL4 mainly functioned in endothelial derived VICs, but not neuro crest derived VICs. Besides, we found the percentage of EdU+, RFP- cells is not increased significantly in Tie2Cre;Vgll4fl/-;RosafsRFP/+ valves compared to Tie2Cre;Vgll4fl/+;RosafsRFP/+ controls( Fig 5C). This means that endothelial deletion of Vgll4 only affect endothelial and endothelial-derived VICs proliferation, but not RFP-( non-endothelial-derived) VICs although they interacted with endothelial, these point enlighten us that Vgll4 function in endothelial derived VICs is cell autonomous effect. VGLL4 total knockout lead to neonatal lethality, while endothelial knockout of VGLL4, heart cushion is normally formed and pups could live till adult stage, this is because in addition to the heart, VGLL4 also widely expressed in many tissues, such as bone, spleen, brain, uterus and so on, so we think the neonatal lethality in Vgll4-/- is a comprehensive defect result, the function of VGLL4 in other organs need to be further investigated. To our knowledge, this is the first in vivo genetic study showing the critical role of the Hippo pathway in regulation of valve remodeling and maturation. From lineage tracing data, we infer that endothelial but not neural crest VGLL4 regulates traced valve interstitial cell proliferation. This could be due to that VGLL4 is mainly expressed in endothelial cells and endothelial derived VIC. Thus, Hippo signaling in VEC is required for post-EMT semilunar valve morphogenesis and homeostasis. Precise regulation of the Hippo pathway is required for proper proliferation of endocardium derived mesenchymal cells and subsequent valve elongation. The present study identifies a previously unknown function of VGLL4 in valve remodeling and homeostasis. These findings suggest that mutations in VGLL4 may underlie human congenital heart valve dysplasia.
VGLL4 , a new member of the Hippo pathway , is intensively investigated in inhibition of tumor progression via competing with YAP to bind TEADs , but its role in cardiovascular field remains unclear . Here we generated VGLL4 knockout mouse line and VGLL4-eGFP reporter mouse line . VGLL4-eGFP reporter mouse line showed VGLL4 was mainly expressed in valve interstitial cells from post-EMT stage to adult stage . Genetic loss of function and lineage tracing data demonstrated only endothelial loss of VGLL4 led to valve malformation with up-regulation of YAP targets . Of note , semi-knockout YAP could rescue this phenotype of VGLL4 knockouts . This is the first study to show the Hippo pathway plays a critical role in valve remodeling , maturation and homeostasis . Our findings suggest that mutations in VGLL4 may underlie human congenital heart valve dysplasia .