Elastogenesis at the onset of human cardiac valve development.
ABSTRACT: Semilunar valve leaflets have a well-described trilaminar histoarchitecture, with a sophisticated elastic fiber network. It was previously proposed that elastin-containing fibers play a subordinate role in early human cardiac valve development; however, this assumption was based on data obtained from mouse models and human second and third trimester tissues. Here, we systematically analyzed tissues from human fetal first (4-12 weeks) and second (13-18 weeks) trimester, adolescent (14-19 years) and adult (50-55 years) hearts to monitor the temporal and spatial distribution of elastic fibers, focusing on semilunar valves. Global expression analyses revealed that the transcription of genes essential for elastic fiber formation starts early within the first trimester. These data were confirmed by quantitative PCR and immunohistochemistry employing antibodies that recognize fibronectin, fibrillin 1, 2 and 3, EMILIN1 and fibulin 4 and 5, which were all expressed at the onset of cardiac cushion formation (~week 4 of development). Tropoelastin/elastin protein expression was first detectable in leaflets of 7-week hearts. We revealed that immature elastic fibers are organized in early human cardiovascular development and that mature elastin-containing fibers first evolve in semilunar valves when blood pressure and heartbeat accelerate. Our findings provide a conceptual framework with the potential to offer novel insights into human cardiac valve development and disease.
Project description:Semilunar valve leaflets have a well-described trilaminar histoarchitecture with a sophisticated elastic fiber network. It was previously proposed that elastin-containing fibers play a subordinate role in early human cardiac valve development; however, this assumption was based on data obtained from mouse models and human second and third trimester tissues. Here, we systematically analyzed tissues from human fetal first (4-12 weeks) and second (13-18 weeks) trimester, adolescent (14-19 years) and adult (50-55 years) hearts to monitor the temporal and spatial distribution of elastic fibers, focusing on semilunar valves. Global gene expression analyses revealed that the transcription of genes essential for elastic fiber formation starts early within the first trimester. These data were confirmed by quantitative PCR and immunohistochemistry employing antibodies that recognize fibronectin, fibrilin-1, -2 and -3, EMILIN-1, fibulin-4 and fibulin-5, which were all expressed at the onset of cardiac cushion formation (~week 4 of development). Tropoelastin/ elastin protein expression was first detectable in leaflets of 7-week hearts. We revealed that immature elastic fibers are organized in early human cardiovascular development, and mature elastin-containing fibers first evolve in semilunar valves when blood pressure and heartbeat accelerate. Our findings provide a conceptual framework with the potential to lead to novel hypotheses in human cardiac valve development and disease. Total RNA obtained from fetal cardiac valve cushions, developed fetal heart valves, adolescent heart valves, and adult heart valves.
Project description:Proper remodeling of the endocardial cushions into thin fibrous valves is essential for gestational progression and long-term function. This process involves dynamic interactions between resident cells and their local environment, much of which is not understood. In this study, we show that deficiency of the cell-cell adhesion protein cadherin-11 (Cad-11) results in significant embryonic and perinatal lethality primarily due to valve related cardiac dysfunction. While endocardial to mesenchymal transformation is not abrogated, mesenchymal cells do not homogeneously cellularize the cushions. These cushions remain thickened with disorganized ECM, resulting in pronounced aortic valve insufficiency. Mice that survive to adulthood maintain thickened and stenotic semilunar valves, but interestingly do not develop calcification. Cad-11 (-/-) aortic valve leaflets contained reduced Sox9 activity, ?1 integrin expression, and RhoA-GTP activity, suggesting that remodeling defects are due to improper migration and/or cellular contraction. Cad-11 deletion or siRNA knockdown reduced migration, eliminated collective migration, and impaired 3D matrix compaction by aortic valve interstitial cells (VIC). Cad-11 depleted cells in culture contained few filopodia, stress fibers, or contact inhibited locomotion. Transfection of Cad-11 depleted cells with constitutively active RhoA restored cell phenotypes. Together, these results identify cadherin-11 mediated adhesive signaling for proper remodeling of the embryonic semilunar valves.
Project description:The elucidation of mechanisms in semilunar valve development might enable the development of new therapies for congenital heart disorders. Here, we found differences in proliferation-associated genes and genes repressed by VEGF between human semilunar valve leaflets from first and second trimester hearts. The proliferation of valve interstitial cells and ventricular valve endothelial cells (VECs) and cellular density declined from the first to the second trimester. Cytoplasmic expression of NFATC1 was detected in VECs (4?weeks) and, later, cells in the leaflet/annulus junction mesenchyme expressing inactive NFATC1 (5.5-9?weeks) were detected, indicative of endocardial-to-mesenchymal transformation (EndMT) in valvulogenesis. At this leaflet/annulus junction, CD44(+) cells clustered during elongation (11?weeks), extending toward the tip along the fibrosal layer in second trimester leaflets. Differing patterns of maturation in the fibrosa and ventricularis were detected via increased fibrosal periostin content, which tracked the presence of the CD44(+) cells in the second trimester. We revealed that spatiotemporal NFATC1 expression actively regulates EndMT during human valvulogenesis, as early as 4?weeks. Additionally, CD44(+) cells play a role in leaflet maturation toward the trilaminar structure, possibly via migration of VECs undergoing EndMT, which subsequently ascend from the leaflet/annulus junction.
Project description:OBJECTIVES:Neither heart valve repair methods nor current prostheses can accommodate patient growth. Normal aortic and pulmonary valves have 3 leaflets, and the goal of valve repair and replacement is typically to restore normal 3-leaflet morphology. However, mammalian venous valves have bileaflet morphology and open and close effectively over a wide range of vessel sizes. We propose that they might serve as a model for pediatric heart valve reconstruction and replacement valve design. We explore this concept using computer simulation. METHODS:We use a finite element method to simulate the ability of a reconstructed cardiac semilunar valve to close competently in a growing vessel, comparing a 3-leaflet design with a 2-leaflet design that mimics a venous valve. Three venous valve designs were simulated to begin to explore the parameter space. RESULTS:Simulations show that for an initial vessel diameter of 12 mm, the venous valve design remains competent as the vessel grows to 20 mm (67%), whereas the normal semilunar design remains competent only to 13 mm (8%). Simulations also suggested that systolic function, estimated as effective orifice area, was not detrimentally affected by the venous valve design, with all 3 venous valve designs exhibiting greater effective orifice area than the semilunar valve design at a given level of vessel growth. CONCLUSIONS:Morphologic features of the venous valve design make it well suited for competent closure over a wide range of vessel sizes, suggesting its use as a model for semilunar valve reconstruction in the growing child.
Project description:Objective: Aortic valve disease is commonly found in the elderly population. It is characterized by dysregulated extracellular matrix remodeling followed by extensive microcalcification of the aortic valve and activation of valve interstitial cells. The mechanism behind these events are largely unknown. Studies have reported expression of hypoxia inducible factor-1 alpha (HIF1?) in calcific nodules in aortic valve disease, therefore we investigated the effect of hypoxia on extracellular matrix remodeling in aged aortic valves. Approach and Results: Western blotting revealed elevated expression of HIF1? and the complex of matrix metalloprotease 9 (MMP9) and neutrophil gelatinase-associated lipocalin (NGAL) in aged porcine aortic valves cultured under hypoxic conditions. Consistently, immunofluorescence staining showed co-expression of MMP9 and NGAL in the fibrosa layer of these porcine hypoxic aortic valves. Gelatinase zymography demonstrated that the activity of MMP9-NGAL complex was significantly increased in aortic valves in 13% O2 compared to 20% O2. Importantly, the presence of ectopic elastic fibers in the fibrosa of hypoxic aortic valves, also detected in human diseased aortic valves, suggests altered elastin homeostasis due to hypoxia. Conclusion: This study demonstrates that hypoxia stimulates pathological extracellular matrix remodeling via expression of NGAL and MMP9 by valve interstitial cells.
Project description:BACKGROUND:The structure of the valve leaflets and sinuses are crucial in supporting the proper function of the semilunar valve and ensuring leaflet durability. Therefore, an enhanced understanding of the structural characteristics of the semilunar valves is fundamental to the evaluation and staging of semilunar valve pathology, as well as the development of prosthetic or bioprosthetic valves. This paper illustrates the process of combining computer-aided design (CAD), 3D printing and flow assessment with 4-dimensional flow magnetic resonance imaging (MRI) to provide detailed assessment of the structural and hemodynamic characteristics of the normal semilunar valve. METHODS:Previously published geometric data on the aortic valve was used to model the 'normal' tricuspid aortic valve with a CAD software package and 3D printed. An MRI compatible flow pump with the capacity to mimic physiological flows was connected to the phantom. A peak flow rate of 100?mL/s and heart rate of 60 beats per minute were used. MRI measurements included cine imaging, 2D and 4D phase-contrast imaging to assess valve motion, flow velocity and complex flow patterns. RESULTS:Cine MRI data showed normal valve function and competency throughout the cardiac cycle in the 3D-printed phantom. Quantitative analysis of 4D Flow data showed net flow through 2D planes proximal and distal to the valve were very consistent (26.03?mL/s and 26.09?mL/s, respectively). Measurements of net flow value agreed closely with the flow waveform provided to the pump (27.74?mL/s), confirming 4D flow acquisition in relation to the pump output. Peak flow values proximal and distal to the valve were 78.4?mL/s and 63.3?mL/s, respectively. Particle traces of flow from 4D-phase contrast MRI data demonstrated flow through the valve into the ascending aorta and vortices within the aortic sinuses, which are expected during ventricular diastole. CONCLUSION:In this proof of concept study, we have demonstrated the ability to generate physiological 3D-printed aortic valve phantoms and evaluate their function with cine- and 4D Flow MRI. This technology can work synergistically with promising tissue engineering research to develop optimal aortic valve replacements, which closely reproduces the complex function of the normal aortic valve.
Project description: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.
Project description:There is limited information about age-specific structural and functional properties of human heart valves, while this information is key to the development and evaluation of living valve replacements for pediatric and adolescent patients. Here, we present an extended data set of structure-function properties of cryopreserved human pulmonary and aortic heart valves, providing age-specific information for living valve replacements. Tissue composition, morphology, mechanical properties, and maturation of leaflets from 16 pairs of structurally unaffected aortic and pulmonary valves of human donors (fetal-53 years) were analyzed. Interestingly, no major differences were observed between the aortic and pulmonary valves. Valve annulus and leaflet dimensions increase throughout life. The typical three-layered leaflet structure is present before birth, but becomes more distinct with age. After birth, cell numbers decrease rapidly, while remaining cells obtain a quiescent phenotype and reside in the ventricularis and spongiosa. With age and maturation-but more pronounced in aortic valves-the matrix shows an increasing amount of collagen and collagen cross-links and a reduction in glycosaminoglycans. These matrix changes correlate with increasing leaflet stiffness with age. Our data provide a new and comprehensive overview of the changes of structure-function properties of fetal to adult human semilunar heart valves that can be used to evaluate and optimize future therapies, such as tissue engineering of heart valves. Changing hemodynamic conditions with age can explain initial changes in matrix composition and consequent mechanical properties, but cannot explain the ongoing changes in valve dimensions and matrix composition at older age.
Project description:Cxcr7-/- mice die a few hours after birth. All of them display semilunar valves abnormalities, including bicuspid aortic or pulmonary valves. Those defects only become obvious before birth. Keywords: genetic modification Overall design: Biological replicates of pools of 3 mutant samples (2 valves with 2-3 leaflets each, so 4-6 leaflets/mutant) and 3 wildtype littermates (6 leaflets/wildtype).
Project description:Atrioventricular heart valves (AHVs) are composed of structurally complex and morphologically heterogeneous leaflets. The coaptation of these leaflets during the cardiac cycle facilitates unidirectional blood flow. Valve regurgitation is treated preferably by surgical repair if possible or replacement based on the disease state of the valve tissue. A comprehensive understanding of valvular morphology and mechanical properties is crucial to refining computational models, serving as a patient-specific diagnostic and surgical tool for preoperative planning. Previous studies have modeled the stress distribution throughout the leaflet's thickness, but validations with layer-specific biaxial mechanical experiments are missing. In this study, we sought to fill this gap in literature by investigating the impact of microstructure constituents on mechanical behavior throughout the thickness of the AHVs' anterior leaflets. Porcine mitral valve anterior leaflets (MVAL) and tricuspid valve anterior leaflets (TVAL) were micro-dissected into three layers (atrialis/spongiosa, fibrosa, and ventricular) and two layers (atrialis/spongiosa and fibrosa/ventricularis), respectively, based on their relative distributions of extracellular matrix components as quantified by histological analyses: collagen, elastin, and glycosaminoglycans. Our results suggest that (i) for both valves, the atrialis/spongiosa layer is the most extensible and anisotropic layer, possibly due to its relatively low collagen content as compared to other layers, (ii) the intact TVAL response is stiffer than the atrialis/spongiosa layer but more compliant than the fibrosa/ventricularis layer, and (iii) the MVAL fibrosa and ventricularis layers behave nearly isotropic. These novel findings emphasize the biomechanical variances throughout the AHV leaflets, and our results could better inform future AHV computational model developments. STATEMENT OF SIGNIFICANCE: This study, which is the first of its kind for atrioventricular heart valve (AHV) leaflet tissue layers, rendered a mechanical characterization of the biaxial mechanical properties and distributions of extracellular matrix components (collagen, elastin, and glycosaminoglycans) of the mitral and tricuspid valve anterior leaflet layers. The novel findings from the present study emphasize the biomechanical variances throughout the thickness of AHV leaflets, and our results indicate that the previously-adopted homogenous leaflet in the AHV biomechanical modeling may be an oversimplification of the complex leaflet anatomy. Such improvement in the understanding of valvular morphology and tissue mechanics is crucial to future refinement of AHV computational models, serving as a patient-specific diagnostic and surgical tool, at the preoperative stage, for treating valvular heart diseases.