The adult mouse heart possesses an extensive lymphatic plexus draining predominantly the subepicardium and the outer layer of the myocardial wall. However, the development of this plexus has not been entirely explored, partially because of the lack of suitable methods for its visualization as well as prolonged lymphatic vessel formation that starts prenatally and proceeds during postnatal stages. Also, neither the course nor location of collecting vessels draining lymph from the mouse heart have been precisely characterized. In this article, we report that murine cardiac lymphatic plexus development that is limited prenatally only to the subepicardial area, postnatally proceeds from the subepicardium toward the myocardial wall with the base-to-apex gradient; this plexus eventually reaches the outer half of the myocardium with a predominant location around branches of coronary arteries and veins. Based on multiple marker immunostaining, the molecular marker-phenotype of cardiac lymphatic endothelial cells can be characterized as: Prox-1 1 , Lyve-1 1 , VEGFR3 1 , Podoplanin 1 , VEGFR2 1 , CD144 1 , Tie2 1 , CD31 1 , vWF 2 , CD34 2 , CD133 2 . There are two major collecting vessels: one draining the right and left ventricles along the left conal vein and running upwards to the left side of the pulmonary trunk and further to the nearest lymph nodes (under the aortic arch and near the trachea), and the other one with its major branch running along the left cardiac vein and further on the surface of the coronary sinus and the left atrium to paratracheal lymph nodes. The extracardiac collectors gain the smooth muscle cell layer during late postnatal stages.

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The role of the cardiac lymphatic system has been recently appreciated since lymphatic disturbances take part in various heart pathologies. This review presents the current knowledge about normal anatomy and structure of lymphatics and their prenatal development for a better understanding of the proper functioning of this system in relation to coronary circulation. Lymphatics of the heart consist of terminal capillaries of various diameters, capillary plexuses that drain continuously subendocardial, myocardial, and subepicardial areas, and draining (collecting) vessels that lead the lymph out of the heart. There are interspecies differences in the distribution of lymphatic capillaries, especially near the valves, as well as differences in the routes and number of draining vessels. In some species, subendocardial areas contain fewer lymphatic capillaries as compared to subepicardial parts of the heart. In all species there is at least one collector vessel draining lymph from the subepicardial plexuses and running along the anterior interventricular septum under the left auricle and further along the pulmonary trunk outside the heart and terminating in the right venous angle. The second collector assumes a different route in various species. In most mammalian species the collectors run along major branches of coronary arteries, have valves and a discontinuous layer of smooth muscle cells.

Chronic wounds are a significant socio-economic problem, thus, the improvement of the effectiveness of their treatment is an important objective for public health strategies. The predominant stage of the chronic wound is the inflammatory reaction which is associated with the damage of tissues, possibly due to the excessive secretion and activation of matrix metalloproteinases (MMPs). Several reports have suggested that amnion dressing inhibits tissue destruction and accelerates wound healing. Our recent study revealed that sterilized amnion stimulates keratinocyte proliferation in vitro, while the present study focused on the clinical application of radiation-sterilized amnion in chronic venous leg ulcers and aimed to explain the possible mechanism of its in vivo action. The study involved 25 individuals suffering from venous leg ulceration with a surface area of 10-100 cm2 and a healing rate below 10% per week, as verified during a 2-week screening period. The effectiveness of the amnion dressing was estimated following 4 weeks of treatment. The wound assessment, based on a modified Bates-Jensen Questionnaire, revealed a good and satisfactory response to the treatment in 23 of the 25 patients. The measurement of MMP-2 and MMP-9 activities in wound exudates revealed a decrease in activity in response to amnion application. This effect resulted from the presence of the potent MMP inhibitors, tissue inhibitor of metalloproteinases-1 (TIMP-1), type-1 plasminogen activator inhibitor (PAI-1) and thrombospondin-1 (TSP-1) in the amnion dressings, as shown by real-time fluorescence zymography and protein microarrays. Thus, unlike modern synthetic dressing materials, radiation-sterilized amnion dressings may have a multidirectional beneficial effect on chronic wounds.

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Vasculogenesis and hematopoiesis are co-localized in the embryonic body, but precise phenotypes of the cells contributing to these processes are not defined. The aim of this study was to characterize phenotypic profiles and location of putative vasculogenic and hematopoietic cellular progenitors in the embryonic mouse heart. Confocal microscopy, as well as ultrastructural and stereomicroscopic analyses, was performed on immunohistochemical whole-mount-stained or sectioned hearts at stages 11.5–14 dpc. A FASC analysis was conducted to quantify putative vasculogenic and hematopoietic cells. We found subepicardial blood islands in the form of foci of accumulation of cells belonging to erythroblastic and megakaryocytic lineages at various stages of maturation, exhibiting phenotypes: GATA2+/CD41+, GATA2−/CD41+, GATA2+/CD71−, GATA2−/CD71+, Fli1+/CD71+, Fli1−/CD71+, with a majority of cells expressing the Ter119 antigen, but none of them expressing Flk1. The subepicardium and the outflow tract endothelium were recognized to be the areas where progenitor cells were scattered or adjoining the endothelial cells. These progenitor cells were characterized as possessing the following antigens: CD45+/Fli1+, CD41+/Flk1+, Flk1+/Fli1+. A FACS analysis demonstrated that the CD41/Flk1 double-positive population of cells constituted 2.68 % of total cell population isolated from 12.5 dpc hearts. Vessels and tubules were positive for CD31, Flk1, Fli1, Tie2, including blood islands endothelia. The endocardial wall endothelia were found to function as an anchoring apparatus for megakaryocytes releasing platelets into the cardiac cavities. Phenotypic characteristics of vasculogenic (Flk1+/Fli1+) and hematopoietic (GATA2+/CD71+, CD41+/GATA2+) progenitors, as well as the putative hemogenic endothelium (Flk1+/CD41+) in embryonic mouse hearts, have been presented. Cardiac blood islands, the subepicardium and endothelium of the outflow tract cushions have been defined as areas where these progenitor cells can be found.

The role of cardiac tissue macrophages (cTMs) during pre- and postnatal developmental stages remains in many aspects unknown. We aimed to characterize cTM populations and their potential functions based on surface markers. Our in situ studies of immunostained cardiac tissue specimens of murine fetuses (from E11to E17) revealed that a significant number of embryonic cTMs (phenotyped by CD45, CD68, CD64, F4/80, CD11b, CD206, Lyve-1) resided mostly in the subepicardial space, not in the entire myocardial wall, as observed in adult individuals. cTMs accompanied newly developed blood and lymphatic vessels adhering to vessel walls by cellular processes. A subpopulation of CD68-positive cells was found to form accumulations in areas of massive apoptosis during the outflow tract remodeling and shortening. Flow cytometry analysis at E14 and E17 stages revealed newly defined three subpopulations:CD64low, CD64highCD206-and CD64highCD206+. The levels of mRNA expression for genes related to regulation of angiogenesis (VEGFa, VEGFb, VEGFc, bFGF), lymphangiogenesis (VEGFc) and extracellular matrix (ECM) remodeling (MMP13, Arg1, Ym1/Chil3, Retlna/FIZZ1) differed among the selected populations and/or embryonic stages. Our results demonstrate a diversity of embryonic cTMs and their tissue-specific locations, suggesting their various potential roles in regulating angiogenesis, lymphangiogenesis and ECM remodeling.

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