Endocardial endothelium in the rat: junctional organization and permeability

Selective permeability of endocardial endothelium has been suggested as a mechanism underlying the modulation of the performance of subjacent myocardium. In this study, we characterized the organization and permeability of junctional complexes in ventricular endocardial endothelium in rat heart. The length of intercellular clefts viewed en face per unit endothelial cell surface area was lower, and intercellular clefts were deeper in endocardial endothelium than in myocardial vascular endothelium, whereas tight junctions had a similar structure in both endothelia. On this basis, endocardia endothelium. might be less permeable than capillary endothelium. However, confocal scanning laser microscopy showed that intravenously injected dextran 10000 coupled to Lucifer Yellow penetrated first the endocardial endothelium and later the myocardial capillary endothelium. Penetration of dextran 10000 in myocardium occurred earlier through subepicardial capillary endothelium than through subendocardial capillary endothelium. Penetration of tracer might thus be influenced by hydrostatic pressure. Dextran of MW 40000 did not diffuse through either endocardial endothelium or capilary endothelium. The ultrastructure of endocardial endothelium may constitute an adaptation to limit diffusion driven by high hydrostatic pressure in the heart. Differences in paracellular diffusion of dextran 10000 between endocardial endothelium and myocardial vessels, may result from differing permeability properties of the endocardium and underlying myocardium.     

Endocardial cells are a distinct endothelial lineage derived from Flk1+ multipotent cardiovascular progenitors

Identification of multipotent cardiac progenitors has provided important insights into the mechanisms of myocardial lineage specification, yet has done little to clarify the origin of the endocardium. Despite its essential role in heart development, characterization of the endocardial lineage has been limited by the lack of specific markers of this early vascular subpopulation. To distinguish endocardium from other vasculature, we generated an NFATc1-nuc-LacZ BAC transgenic mouse line capable of labeling this specific endothelial subpopulation at the earliest stages of cardiac development. To further characterize endocardiogenesis, embryonic stem cells (ESCs) derived from NFATc1-nuc-LacZ blastocysts were utilized to demonstrate that endocardial differentiation in vitro recapitulates the close temporal–spatial relationship observed between myocardium and endocardium seen in vivo. Endocardium is specified as a cardiac cell lineage, independent from other vascular populations, responding to BMP and Wnt signals that enhance cardiomyocyte differentiation. Furthermore, a population of Flk1+ cardiovascular progenitors, distinct from hemangioblast precursors, represents a mesodermal precursor of the endocardial endothelium, as well as other cardiovascular lineages. Taken together, these studies emphasize that the endocardium is a unique cardiac lineage and provides further evidence that endocardium and myocardium are derived from a common precursor.     

TELOCYTES IN ENDOCARDIUM: Electron Microscope Evidence

The term TELOCYTES was very recently introduced, for replacing the name Interstitial Cajal‐Like Cells (ICLC). In fact, telocytes are not really Cajal‐like cells, they being different from all other interstitial cells by the presence of telopodes, which are cell‐body prolongations, very thin (under the resolving power of light microscopy), extremely long (tens up to hundreds of micrometers), with a moniliform aspect (many dilations along), and having caveolae. The presence of telocytes in epicardium and myocardium was previously documented. We present here electron microscope images showing the existence of telocytes, with telopodes, at the level of mouse endocardium. Telocytes are located in the subendothelial layer of endocardium, and their telopodes are interposed in between the endocardial endothelium and the cardiomyocytes bundles. Some telopodes penetrate from the endocardium among the cardiomyocytes and surround them, eventually. Telopodes frequently establish close spatial relationships with myocardial blood capillaries and nerve endings. Because we may consider endocardium as a ‘blood–heart barrier’, or more exactly as a ‘blood–myocardium barrier’, telocytes might have an important role in such a barrier being the dominant cell population in subendothelial layer of endocardium.     

Possible sites of ultrafiltration in Tubifex tubifex Müller (Annelida,Oligochaeta)

Summary The endothelia of Tubifex tubifex Müller consist of myoendothelial cells, chloragocytes, or podocytes. The latter seem to occur only as windows on the ventral vessel which has an endothelium of myoendothelial cells elsewhere. The podocytes are large cells, with several processes on the inner side which ramify into several pedicels. These are aligned upon the outside of the basement membrane which lines the inside of the endothelium. The gaps between adjacent pedicels are about 40 nm wide. In capillaries fenestrated endothelia occur with irregular spacings measuring up to 0.4–1 m. A diaphragm in podocytes or capillary fenestrations do not seem to exist. The basement membrane is the only continuous layer lining the blood vessels and capillaries of Tubifex with a rather uniform diameter in the range of 50 nm. It is the only permeability barrier between blood and coelomic fluid.     

Endocardial surface and atrial morphological changes during development and aging

Light, scanning, and transmission electron microscopic observations related to morphological changes of the right atrium as well as the atrial endocardium during development (15th embryonic day and 1 day old) and aging (560 days old) in the Syrian hamster were described and correlated. From the fetus to the adult, the atrial endocardium differentiates in parallel with, or in response to, the subjacent proliferating myocytes in the atrial wall and the trabeculae. Simultaneously, the atrium compartmentalizes grossly into a main chamber and an appendicular region. There is a progressive differentiation from a rudimentary, open chamber with primitive mural ridges in the fetal atria to a distinct, separate, atrial main chamber and appendage with a dense network of trabeculae in the adult. The fetal and neonatal endocardial, endothelial cells are convex with a central nuclear bulging and attenuated cytoplasmic extensions; the adult endocardium shows a squamous endothelium. Two cell surface specializations were observed in all age groups: microvilli and blebs or cytoplasmic protrusions. The general atrial morphology and surface endocardial changes were correlated with growth and the role of the endocardial endothelium as a barrier which controls metabolic exchanges, including the transport of atrial natriuretic factor, between the myocytes and the blood. This endothelial function appears to be essential in the fetal and neonatal age groups since no blood vessels are detected in these groups.     

Differences in regional capillary distribution and myocyte sizes in normal and hypertrophic rat hearts.

Data are reported which show significant regional capillary differences in left ventricular endocardium and epicardium of normal rats and of rats with hyperthyroid-induced cardiac hypertrophy. The epicardial region of control rats has 38% more capillaries than the endocardial region. Control endocardial myocytes are 62% larger in cross-sectional area than epicardial myocytes. Hypertrophic hearts exhibit regional differences in capillary density similar to those in the normal hearts, but there is an overall reduction of 12 and 17.5% in capillary density in both regions. The average cross-sectional area of myocytes increases 34.5% in the epicardium and 22.5% in the endocardium.     

Interstitial cells from the atrial and ventricular sides of the bovine mitral valve respond differently to denuding endocardial injury

Summary The mitral valve has atrial and ventricular sides, each lined by endocardial cells. The valve stroma contains α smooth muscle actin positive interstitial cells, collagen, glycosaminoglycans, and elastic tissue. To eliminate the effect of endocardium on wound repair in bovine mitral valve organ culture, the endocardium was removed from both sides of the valve. At 6 days, organ cultures of these preparations revealed surface cells on the ventricular side but not on the atrial side. Ventricular surface cells were negative for Factor VIII-related antigen, and positive for α smooth muscle actin. Immuno-peroxidase staining for proliferating cell nuclear antigen/cyclin, a marker for cell proliferation, revealed a positive labeling index of (mean ± standard deviation) 0.08 ± 0.16% for interstitial cells from the atrial side and 0.14 ± 0.19% for ventricular side interstitial cells in uncultured preparations (not significant), and 0.44 ± 0.69% for atrial side interstitial cells and 2.25 ± 1.64% for ventricular side interstitial cells in the cultured preparations (significant,P<0.0006). The results suggest that in organ culture, interstitial cells from the ventricular side of the mitral valve respond to a denuding endocardial injury by proliferating and migrating onto the adjacent surface whereas interstitial cells from the atrial side do not. This difference in the response to injury of interstitial cells from the atrial and ventricular sides of the valve may reflect differences in phenotype or may be due to effects of extracellular matrix on interstitial cell behavior. The latter is possible because of differences in the extracellular matrix of the atrial and ventricular sides of the valve.     

Quantitative immunocytochemical studies of endogenous albumin in rat aortic endothelial and mesothelial cells

Endogenous albumin was revealed over cellular structures of rat ascendent aorta endothelia and mesothelium, with high resolution and specificity, by applying the protein A-gold immunocytochemical approach. This approach allows albumin distribution to be studied under steady-state conditions. The cellular layers evaluated were the aortic endothelium, the capillary endothelium (vasa vasorum), and the mesothelium externally lining the aorta at this level. Gold particles, revealing albumin antigenic sites, were preferentially located over plasmalemmal vesicles and intercellular clefts of endothelial and mesothelial cells, though with different labeling intensities. The interstitial space was also labeled. Morphometrical evaluation of plasmalemmal vesicles demonstrated a higher surface density for these structures in capillary endothelial cells (12%) compared with those in aortic endothelial (5%) and mesothelial cells (2%). Quantitation of gold labeling intensities over these structures revealed a higher labeling over plasmalemmal vesicles of capillary endothelium than over those of aortic endothelium and mesothelium. This result, together with the higher surface density of plasmalemmal vesicles found in capillary endothelium, suggest an important role of these structures in the transendothelial passage of endogenous albumin, particularly for capillary endothelium. On the other hand, labeling densities over mesothelial clefts were found to be higher than those of capillary and aortic endothelia. Results from this study concur with the proposal of a differential passage of albumin according to the cell lining considered, and suggest to a role for mesothelial intercellular clefts in contributing to the presence of albumin in interstitial spaces.     

Endothelial cell lineages of the heart

During early gastrulation, vertebrate embryos begin to produce endothelial cells (ECs) from the mesoderm. ECs first form primitive vascular plexus de novo and later differentiate into arterial, venous, capillary, and lymphatic ECs. In the heart, the five distinct EC types (endocardial, coronary arterial, venous, capillary, and lymphatic) have distinct phenotypes. For example, coronary ECs establish a typical vessel network throughout the myocardium, whereas endocardial ECs form a large epithelial sheet with no angiogenic sprouting into the myocardium. Neither coronary arteries, veins, and capillaries, nor lymphatic vessels fuse with the endocardium or open to the heart chamber. The developmental stage during which the specific phenotype of each cardiac EC type is determined remains unclear. The mechanisms involved in EC commitment and diversity can however be more precisely defined by tracking the migratory patterns and lineage decisions of the precursors of cardiac ECs. Work carried out by the authors is supported in part by the NIH.     

Endocardial endothelium in the rat: cell shape and organization of the cytoskeleton

The cytoskeleton in endocardial endothelium of rat heart was examined by en face confocal scanning laser microscopy. In the ventricular cavity, endocardial endothelial cells had a polygonal shape and F-actin staining was generally restricted to the peripheral junctional actin band. Central F-actin bundles, or stress fibers, in endocardial endothelial cells were found on the tendon end of papillary muscles, especially in the right ventricle, and frequently in the outflow tract of both ventricles; elsewhere, stress fibers were scarce. Many endocardial endothelial cells were elongated in areas of endothelium with stress fibers, but no correlation was found between cell elongation and the number of stress fibers. An inverse correlation was found between the number of stress fibers and the surface area of endocardial endothelial cells. Shear stress as well as mechanical deformation of the surface of the ventricular wall during the cardiac cycle may affect cell shape and the organization of actin filaments in endocardial endothelial cells. Vimentin in endocardial endothelial cells formed a filamentous network with some distinct cytoplasmic and juxtanuclear vimentin bundles. No perinuclear ring of vimentin filaments was observed in endocardial endothelium. Microtubules in endocardial endothelial cells were, in contrast to endothelial cells of rat aorta, not aligned, less closely packed and originated from randomly distributed centriolar regions. The cytoskeleton has been suggested to play an important role in cellular functions of vascular endothelial cells. Accordingly, differences in the cytoskeletal organization between endocardial and vascular endothelial cells may relate to differences in functional properties.     

Signaling via the Tgf-beta type I receptor Alk5 in heart development

Trophic factors secreted both from the endocardium and epicardium regulate appropriate growth of the myocardium during cardiac development. Epicardially-derived cells play also a key role in development of the coronary vasculature. This process involves transformation of epithelial (epicardial) cells to mesenchymal cells (EMT). Similarly, a subset of endocardial cells undergoes EMT to form the mesenchyme of endocardial cushions, which function as primordia for developing valves and septa. While it has been suggested that transforming growth factor-βs (Tgf-β) play an important role in induction of EMT in the avian epi- and endocardium, the function of Tgf-βs in corresponding mammalian tissues is still poorly understood. In this study, we have ablated the Tgf-β type I receptor Alk5 in endo-, myo- and epicardial lineages using the Tie2-Cre, Nkx2.5-Cre, and Gata5-Cre driver lines, respectively. We show that while Alk5-mediated signaling does not play a major role in the myocardium during mouse cardiac development, it is critically important in the endocardium for induction of EMT both in vitro and in vivo. Moreover, loss of epicardial Alk5-mediated signaling leads to disruption of cell-cell interactions between the epicardium and myocardium resulting in a thinned myocardium. Furthermore, epicardial cells lacking Alk5 fail to undergo Tgf-β-induced EMT in vitro. Late term mutant embryos lacking epicardial Alk5 display defective formation of a smooth muscle cell layer around coronary arteries, and aberrant formation of capillary vessels in the myocardium suggesting that Alk5 is controlling vascular homeostasis during cardiogenesis. To conclude, Tgf-β signaling via Alk5 is not required in myocardial cells during mammalian cardiac development, but plays an irreplaceable cell-autonomous role regulating cellular communication, differentiation and proliferation in endocardial and epicardial cells.     

Tubular invaginations in cerebral endothelium and their relation to smooth-surfaced cisternae in hagfish (Myxine glutinosa)

Summary The organization of vesicular profiles in the endothelium of cerebral capillaries of the hagfish, Myxine glutinosa, has been reinvestigated. Judged from random thin sections the endothelial cells contain numerous vesicles and tubules, in contrast to brain endothelia of most other vertebrates. However, three-dimensional reconstructions based on ultrathin serial sections (thickness 18 nm) showed that the profiles represent a system of irregular tubular invaginations of the cell membrane, comparable to the vesicular invaginations demonstrated in extracerebral capillary endothelia of frogs and rats. In addition, smooth-surfaced cisternae were present in close relation to the invaginations. The function of endothelial invaginations is unknown. They do not transport macromolecules, because the blood-brain barrier is practically impermeable to proteins. However, since the system of the invaginations and smooth-surfaced cisternae is structurally similar to the system of caveolae and sarcoplasmic reticulum in smooth muscle cells, a common function seems likely. It is proposed that endothelial invaginations and smooth-surfaced cisternae are involved in regulation of cytosolic Ca⁺⁺-concentration.     

The heart endocardium is derived from vascular endothelial progenitors

The embryonic heart is composed of two cell layers: the myocardium, which contributes to cardiac muscle tissue, and the endocardium, which covers the inner lumen of the heart. Whereas significant progress has been made toward elucidating the embryonic origins of the myocardium, the origins of the endocardium remain unclear. Here, we have identified an endocardium-forming field medial to the cardiac crescent, in a continuum with the endothelial plexus. In vivo live imaging of quail embryos revealed that endothelial progenitors, like second/anterior heart field progenitors, migrate to, and enter, the heart from the arterial pole. Furthermore, embryonic endothelial cells implanted into the cardiac crescent contribute to the endocardium, but not to the myocardium. In mouse, lineage analysis focusing on endocardial cells revealed an unexpected heterogeneity in the origins of the endocardium. To gain deeper insight into this heterogeneity, we conditionally ablated Flk1 in distinct cardiovascular progenitor populations; FLK1 is required in vivo for formation of the endocardium in the Mesp1 and Tie2 lineages, but not in the Isl1 lineage. Ablation of Flk1 coupled with lineage analysis in the Isl1 lineage revealed that endothelium-derived Isl1(-) endocardial cells were significantly increased, whereas Isl1(+) endocardial cells were reduced, suggesting that the endocardium is capable of undergoing regulative compensatory growth. Collectively, our findings demonstrate that the second heart field contains distinct myocardial and endocardial progenitor populations. We suggest that the endocardium derives, at least in part, from vascular endothelial cells.     

Invasion of mesenchyme into three-dimensional collagen gels: a regional and temporal analysis of interaction in embryonic heart tissue

In normal heart development the endothelium of the atrioventricular canal, but not the ventricle, produces mesenchymal cells which seed (invade) into the intervening extracellular matrix toward the myocardium at around 64-69 hr of development. We have utilized three-dimensional collagen substrates to examine the initiation of seeding by atrioventricular canal endothelia in vitro and to compare and contrast the responses of the ventricular endothelia. Explants of atrioventricular canals and ventricles from staged embryos were placed on the surfaces of collagen gels prior to the onset of seeding in situ. At varied intervals of incubation, the explant was removed, leaving behind a monolayer on the surface of the gel which consisted of endothelial cells. Subsequently, the endothelial outgrowths were examined for seeded cells. The results confirm the regional endothelial differences seen in vivo. They also show that invasion of the collagen gels is due to an alteration in phenotype mediated by interaction with other components of embryonic heart explant. Lastly, the time course of this tissue interaction in vitro mimics the onset of seeding in vivo.     

Epithelial-mesenchymal transitions in the developing heart of the dogfish (Scyliorhinus canicula). A scanning electron microscopic study

An epithelial-mesenchymal transition is involved in two main morphogenetic events of cardiac morphogenesis, namely the differentiation of the valvuloseptal tissue from the endocardial endothelium, and the formation of subepicardial mesenchyme from the epicardial mesothelium. We have proposed that the dogfish ( Scyliorhinus canicula ) is a suitable model for the study of basic processes of cardiac morphogenesis in vertebrates, since the heart of this primitive fish probably outlines the original bauplan of the vertebrate heart. In order to study in this model the endocardial and epicardial epithelial-mesenchymal transition under scanning electron microscopy, we have used a technique of paraffin-embedding, partial sectioning, dewaxing and critical-point drying. Our results showed: 1) A centrifugal pattern of epicardial development from the atrioventricular groove to the sinus venosus and conus arteriosus; 2) A close spatial and temporal relationship between the endocardial and epicardial epithelial-mesenchymal transition, although the transformation of the endocardium starts earlier and ends later the epicardial transformation; 3) A complex arrangement of the fibrous extracellular matrix which is established prior to the migration of the mesenchymal cells. Subepicardial, but not subendothelial mesenchymal cells, coalesce in unicellular or pluricellular ring-like structures that probably are related to the origin of the cardiac vessels.     

Expression of Crip2, a LIM-domain-only protein, in the mouse cardiovascular system under physiological and pathological conditions

LIM domain-containing proteins mediate protein–protein interactions and play regulatory roles in various physiopathological processes. The mRNA of Crip2, a LIM-only gene, has been detected abundantly in developing and adult hearts but its cell-type specific expression profile has not been well characterized. In this study, we showed that Crip2 is highly expressed in the myocardium, moderately expressed in the endocardium and absent from the epicardium of the developing mouse heart. Interestingly, Crip2 expression is present in the endocardial cells that line both endocardial cushions, whereas it is markedly reduced in the cushion mesenchymes during valve leaflet formation. In the developing vascular system, Crip2 is detected in the endothelial cells of both blood and lymphatic vessels. Consistent with the expression pattern observed in embryos, Crip2 is also highly expressed in the myocardium, endocardium and coronary vascular endothelial cells of the adult heart. In the cardiomyocytes, Crip2 is colocalized with cardiac troponin T in the thin-filaments of sarcomeres. Nonetheless, experimental studies revealed that the expression level of Crip2 is not altered in the isoproterenol (ISO) induced hypertrophic heart. Moreover, Crip2 is detected in endothelial cells of the neovasculature during wound healing and tumor growth. The persistence of Crip2 expression in cardiovascular tissues implies that Crip2 might exert an important impact on the cardiovascular development, maintenance and homeostasis.     

Origins and patterning of avian outflow tract endocardium

Outflow tract endocardium links the atrioventricular lining, which develops from cardiogenic plate mesoderm, with aortic arches, whose lining forms collectively from splanchnopleuric endothelial channels, local endothelial vesicles, and invasive angioblasts. At two discrete sites, outflow tract endocardial cells participate in morphogenetic events not within the repertoire of neighboring endocardium: they form mesenchymal precursors of endocardial cushions. The objectives of this research were to document the history of outflow tract endocardium in the avian embryo immediately prior to development of the heart, and to ascertain which, if any, aspects of this history are necessary to acquire cushion-forming potential. Paraxial and lateral mesodermal tissues from between somitomere 3 (midbrain level) and somite 5 were grafted from quail into chick embryos at 3-10 somite stages and, after 2-5 days incubation, survivors were fixed and sectioned. Tissues were stained with the Feulgen reaction to visualize the quail nuclear marker or with antibodies (monoclonal QH1 or polyclonals) that recognize quail but not chick cells. Many quail endothelial cells lose the characteristic nuclear heterochromatin marker, but they retain the species-specific epitope recognized by these antibodies. Precursors of outflow tract but not atrioventricular endocardium are present in cephalic paraxial and lateral mesoderm, with their greatest concentration at the level of the otic placode. Furthermore, the ventral movement of individual angiogenic cells is a normal antecedent to outflow tract formation. Cardiac myocytes were never derived from grafted head mesoderm. Thus, unlike the atrioventricular regions of the heart, outflow tract endocardial and myocardial precursors do not share a congruent embryonic history. The results of heterotopic transplantation, in which trunk paraxial or lateral mesoderm was grafted into the head, were identical, including the formation of cushion mesenchyme. This means that cushion positioning and inductive influences must operate locally within the developing heart tubes.     

Intraorganic lymphatic bed of the cattle heart

Lymphomicrocirculatory networks of endocardium, myocardium and epicardium, as well as lymphatic vessels of four orders represent the intraorganic lymphatic bed of the cattle heart. In the endocardium there is a lymphatic network with close loops and a small amount of blindly beginning capillaries. The capillary lymphatic bed of the endocardial trabeculae carneae is much more dense than that in the other part of the endocardial surface. The spatial lymphatic network of the myocardium is joined with the lymphomicrocirculatory networks of the endocardium and epicardium by means of a large amount of connections. The epicardial lymphatic bed is formed by blindly beginning lymphatic capillaries, which situate in close and nonclose loops of the lymphatic network. In the epicardium there is only one lymphatic network. The size of the loops and the diameter of the lymphatic capillaries is directly proportional to the age of the animals.