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.

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.

Cardiomyocyte-fibroblast crosstalk in the postnatal heart.

During the postnatal period in mammals, the heart undergoes significant remodeling in response to increased circulatory demands. In the days after birth, cardiac cells, including cardiomyocytes and fibroblasts, progressively lose embryonic characteristics concomitant with the loss of the heart's ability to regenerate. Moreover, postnatal cardiomyocytes undergo binucleation and cell cycle arrest with induction of hypertrophic growth, while cardiac fibroblasts proliferate and produce extracellular matrix (ECM) that transitions from components that support cellular maturation to production of the mature fibrous skeleton of the heart. Recent studies have implicated interactions of cardiac fibroblasts and cardiomyocytes within the maturing ECM environment to promote heart maturation in the postnatal period. Here, we review the relationships of different cardiac cell types and the ECM as the heart undergoes both structural and functional changes during development. Recent advances in the field, particularly in several recently published transcriptomic datasets, have highlighted specific signaling mechanisms that underlie cellular maturation and demonstrated the biomechanical interdependence of cardiac fibroblast and cardiomyocyte maturation. There is increasing evidence that postnatal heart development in mammals is dependent on particular ECM components and that resulting changes in biomechanics influence cell maturation. These advances, in definition of cardiac fibroblast heterogeneity and function in relation to cardiomyocyte maturation and the extracellular environment provide, support for complex cell crosstalk in the postnatal heart with implications for heart regeneration and disease mechanisms.

Stem Cell-Secreted Allogeneic Elastin-Rich Matrix with Subsequent Decellularization for the Treatment of Critical Valve Diseases in the Young.

Critical valve diseases in infants have a very poor prognosis for survival. Particularly challenging is for the valve replacement to support somatic growth. From a valve regenerative standpoint, bio-scaffolds have been extensively investigated recently. While bio-scaffold valves facilitate acute valve functionality, their xenogeneic properties eventually induce a hostile immune response. Our goal was to investigate if a bio-scaffold valve could be deposited with tissues derived from allogeneic stem cells, with a specific dynamic culture protocol to enhance the extracellular matrix (ECM) constituents, with subsequent stem cell removal. Porcine small intestinal submucosa (PSIS) tubular-shaped bio-scaffold valves were seeded with human bone marrow-derived mesenchymal stem cells (hBMMSCs), cultured statically for 8 days, and then exposed to oscillatory fluid-induced shear stresses for two weeks. The valves were then safely decellularized to remove the hBMMSCs while retaining their secreted ECM. This de novo ECM was found to include significantly higher (p < 0.05) levels of elastin compared to the ECM produced by the hBMMSCs under standard rotisserie culture. The elastin-rich valves consisted of ~8% elastin compared to the ~10% elastin composition of native heart valves. Allogeneic elastin promotes chemotaxis thereby accelerating regeneration and can support somatic growth by rapidly integrating with the host following implantation. As a proof-of-concept of accelerated regeneration, we found that valve interstitial cells (VICs) secreted significantly more (p < 0.05) collagen on the elastin-rich matrix compared to the raw PSIS bio-scaffold.

De Novo Valve Tissue Morphology Following Bioscaffold Mitral Valve Replacement in a Juvenile Non-Human Primate Model.

The utility of implanting a bioscaffold mitral valve consisting of porcine small intestinal submucosa (PSIS) in a juvenile baboon model (12 to 14 months old at the time of implant; n = 3) to assess their in vivo tissue remodeling responses was investigated. Our findings demonstrated that the PSIS mitral valve exhibited the robust presence of de novo extracellular matrix (ECM) at all explantation time points (at 3-, 11-, and 20-months). Apart from a significantly lower level of proteoglycans in the implanted valve's annulus region (p < 0.05) at 3 months compared to the 11- and 20-month explants, there were no other significant differences (p > 0.05) found between any of the other principal valve ECM components (collagen and elastin) at the leaflet, annulus, or chordae tendinea locations, across these time points. In particular, neochordae tissue had formed, which seamlessly integrated with the native papillary muscles. However, additional processing will be required to trigger accelerated, uniform and complete valve ECM formation in the recipient. Regardless of the specific processing done to the bioscaffold valve, in this proof-of-concept study, we estimate that a 3-month window following bioscaffold valve replacement is the timeline in which complete regeneration of the valve and integration with the host needs to occur.

Physiologically Relevant Fluid-Induced Oscillatory Shear Stress Stimulation of Mesenchymal Stem Cells Enhances the Engineered Valve Matrix Phenotype.

Support of somatic growth is a fundamental requirement of tissue-engineered valves. However, efforts thus far have been unable to maintain this support long term. A key event that will determine the valve's long-term success is the extent to which healthy host tissue remodeling can occur on the valve soon after implantation. The construct's phenotypic-status plays a critical role in accelerating tissue remodeling and engineered valve integration with the host via chemotaxis. In the current study, human bone-marrow-derived mesenchymal stem cells were utilized to seed synthetic, biodegradable scaffolds for a period of 8 days in rotisserie culture. Subsequently, cell-seeded scaffolds were exposed to physiologically relevant oscillatory shear stresses (overall mean, time-averaged shear stress, ~7.9 dynes/cm2; overall mean, oscillatory shear index, ~0.18) for an additional 2 weeks. The constructs were found to exhibit relatively augmented endothelial cell expression (CD31; compared to static controls) but concomitantly served to restrict the level of the activated smooth muscle phenotype (α-SMA) and also produced very low stem cell secretion levels of fibronectin (p < 0.05 compared to static and rotisserie controls). These findings suggest that fluid-induced oscillatory shear stresses alone are important in regulating a healthy valve phenotype of the engineered tissue matrix. Moreover, as solid stresses could lead to increased α-SMA levels, they should be excluded from conditioning during the culture process owing to their associated potential risks with pathological tissue remodeling. In conclusion, engineered valve tissues derived from mesenchymal stem cells revealed both a relatively robust valvular phenotype after exposure to physiologically relevant scales of oscillatory shear stress and may thereby serve to accelerate healthy valve tissue remodeling in the host post-implantation.

Uncovering the Mechanical, Thermal, and Chemical Characteristics of Biodegradable Mushroom Leather with Intrinsic Antifungal and Antibacterial Properties.

The growing demand for a sustainable leather industry with a low environmental impact has prompted the development of alternative vegetable-based materials. In this study, a biodegradable mushroom-based leather derived from the fruiting body of Phellinus ellipsoideus is investigated. The biodegradable leather proves to be thermally stable up to 250 °C. The mechanically robust macrostructure combines a tensile strength of 1.2 MPa and ductility (101% strain at break) attributed to the natural balance of chitin (0.3) and proteins (0.7) constituting the mycelium fibers. The chitin-protein system results in an intrinsic scratch-resistant structure with exciting damping properties in a low frequency range. Enhanced damping capabilities within 5-20 Hz (tan δ: 0.1-0.20) are attributed to the macrostuctural alignment of the mycelium under cyclic tension. Whereas, increasing frequencies >20 Hz induce micromolecular interactions between chitin and proteins within the fibers. Exposure of the bioleather to acidic (pH 4, 5) and basic (pH 8, 9) media demonstrated the selective dissolution of proteins (basic) and chitin (acid) components within the mycelium, opening an opportunity for tunable mechanical response. Reducing the protein content induced an increase in stiffness and strength (pH 8 and 9), while reducing its chitin component showed variable ductility (pH 4 and 5). Owing to the entirely natural composition of the mushroom leather, intrinsic antifungal and antibacterial properties found in the mycelium resist fungal invasion and bacterial growth. Thus, this study displays the unique morphology-property relationship of a biodegradable mushroom leather, proving its potential as a fully sustainable and environmentally friendly alternative.

Porcine Small Intestinal Submucosa Mitral Valve Material Responses Support Acute Somatic Growth.

Background: Conceptually, a tissue engineered heart valve would be especially appealing in the pediatric setting since small size and somatic growth constraints would be alleviated. In this study, we utilized porcine small intestinal submucosa (PSIS) for valve replacement. Of note, we evaluated the material responses of PSIS and subsequently its acute function and somatic growth potential in the mitral position. Methods and Results: Material and mechanical assessment demonstrated that both fatigued 2ply (∼65 µm) and 4ply (∼110 µm) PSIS specimens exhibited similar failure mechanisms, but at an accelerated rate in the former. Specifically, the fatigued 2ply PSIS samples underwent noticeable fiber pullout and recruitment on the bioscaffold surface, leading to higher yield strength (p < 0.05) and yield strain (p < 0.05) compared to its fatigued 4ply counterparts. Consequently, 2ply PSIS mitral valve constructs were subsequently implanted in juvenile baboons (n = 3). Valve function was longitudinally monitored for 90 days postvalve implantation and was found to be robust in all animals. Histology at 90 days in one of the animals revealed the presence of residual porcine cells, fibrin matrix, and host baboon immune cells but an absence of tissue regeneration. Conclusions: Our findings suggest that the altered structural responses of PSIS, postfatigue, rather than de novo tissue formation, are primarily responsible for the valve's ability to accommodate somatic growth during the acute phase (90 days) following mitral valve replacement. Impact Statement Tissue engineered heart valves (TEHVs) offer the potential of supporting somatic growth. In this study, we investigated a porcine small intestinal submucosa bioscaffold for pediatric mitral heart valve replacement. The novelty of the study lies in identifying material responses under mechanical loading conditions and its effectiveness in being able to function as a TEHV. In addition, the ability of the scaffold valve to support acute somatic growth was evaluated in the Baboon model. The current study contributes toward finding a solution for critical valve diseases in children, whose current prognosis for survival is poor.

Elastin-Dependent Aortic Heart Valve Leaflet Curvature Changes During Cyclic Flexure.

The progression of calcific aortic valve disease (CAVD) is characterized by extracellular matrix (ECM) remodeling, leading to structural abnormalities and improper valve function. The focus of the present study was to relate aortic valve leaflet axial curvature changes as a function of elastin degradation, which has been associated with CAVD. Circumferential rectangular strips (L × W = 10 × 2.5 mm) of normal and elastin-degraded (via enzymatic digestion) porcine AV leaflets were subjected to cyclic flexure (1 Hz). A significant increase in mean curvature (p < 0.05) was found in elastin-degraded leaflet specimens in comparison to un-degraded controls at both the semi-constrained (50% of maximum flexed state during specimen bending and straightening events) and fully-constrained (maximally-flexed) states. This significance did not occur in all three flexed configurations when measurements were performed using either minimum or maximum curvature. Moreover, the mean curvature increase in the elastin-degraded leaflets was most pronounced at the instance of maximum flexure, compared to un-degraded controls. We conclude that the mean axial curvature metric can detect distinct spatial changes in aortic valve ECM arising from the loss in bulk content and/or structure of elastin, particularly when there is a high degree of tissue bending. Therefore, the instance of maximum leaflet flexure during the cardiac cycle could be targeted for mean curvature measurements and serve as a potential biomarker for elastin degradation in early CAVD remodeling.