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.