The proepicardium delivers hemangioblasts but not lymphangioblasts to the developing heart

The mass of the myocardium and endocardium of the vertebrate heart derive from the heart-forming fields of the lateral plate mesoderm. Further components of the mature heart such as the epicardium, cardiac interstitium and coronary blood vessels originate from a primarily extracardiac progenitor cell population: the proepicardium (PE). The coronary blood vessels are accompanied by lymph vessels, suggesting a common origin of the two vessel types. However, the origin of cardiac lymphatics has not been studied yet. We have grafted PE of HH-stage 17 (day 3) quail embryos hetero- and homotopically into chick embryos, which were re-incubated until day 15. Double staining with the quail endothelial cell (EC) marker QH1 and the lymphendothelial marker Prox1 shows that the PE of avian embryos delivers hemangioblasts but not lymphangioblasts. We have never observed quail ECs in lymphatics of the chick host. However, one exception was a large lymphatic trunk at the base of the chick heart, indicating a lympho-venous anastomosis and a 'homing' mechanism of venous ECs into the lymphatic trunk. Cardiac lymphatics grow from the base toward the apex of the heart. In murine embryos, we observed a basal to apical gradient of scattered Lyve-1+/CD31+/CD45+ cells in the subepicardium at embryonic day 12.5, indicating a contribution of immigrating lymphangioblasts to the cardiac lymphatic system. Our studies show that coronary blood and lymph vessels are derived from different sources, but grow in close association with each other.     

New insights into the plasticity of the endothelial phenotype

The mammalian vascular system consists of two distinct, but closely related, networks: the blood vasculature (itself divided into arterial and venous networks) and the lymphatic vasculature. EC (endothelial cell) lineage specification has been proposed to be determined during embryonic development, after which the ECs are committed to their fate. However, increasing evidence suggests that ECs retain various degrees of plasticity, and have the ability to express characteristics of alternative cell lineages. Therapeutic control of endothelial plasticity will allow greater understanding of the genesis and treatment of several vascular diseases.     

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.     

Subepicardial endothelial cells invade the embryonic ventricle wall to form coronary arteries

Coronary arteries bring blood flow to the heart muscle. Understanding the developmental program of the coronary arteries provides insights into the treatment of coronary artery diseases. Multiple sources have been described as contributing to coronary arteries including the proepicardium, sinus venosus (SV), and endocardium. However, the developmental origins of coronary vessels are still under intense study. We have produced a new genetic tool for studying coronary development, an AplnCreER mouse line, which expresses an inducible Cre recombinase specifically in developing coronary vessels. Quantitative analysis of coronary development and timed induction of AplnCreER fate tracing showed that the progenies of subepicardial endothelial cells (ECs) both invade the compact myocardium to form coronary arteries and remain on the surface to produce veins. We found that these subepicardial ECs are the major sources of intramyocardial coronary vessels in the developing heart. In vitro explant assays indicate that the majority of these subepicardial ECs arise from endocardium of the SV and atrium, but not from ventricular endocardium. Clonal analysis of Apln-positive cells indicates that a single subepicardial EC contributes equally to both coronary arteries and veins. Collectively, these data suggested that subepicardial ECs are the major source of intramyocardial coronary arteries in the ventricle wall, and that coronary arteries and veins have a common origin in the developing heart.     

Dynamic changes in endoglin expression in the developing mouse heart

Endoglin (ENG) is essential for cardiovascular development and is expressed in the heart from its earliest developmental stages. ENG expression has been reported in the cardiac crescent, endocardium, valve mesenchyme and coronary vascular endothelial cells. However, its expression in these cell types is non-uniform and the dynamic changes in ENG expression during heart development have not been systematically studied.Using immunofluorescent staining we tracked ENG protein expression in mouse embryonic hearts aged from 11.5 to 17.5 days, and in postnatal and adult hearts. ENG is expressed in the endocardium and in venous endothelial cells throughout these developmental stages. ENG protein is down-regulated by approximately two-fold as a subset of early coronary veins reprogram to form arteries within the developing myocardium from E13.5. This two-fold higher ratio of ENG protein in veins versus arteries is maintained throughout cardiac development and in the adult heart.ENG is also down-regulated two-fold following mesenchymal transition of endocardial cells to form cardiac valve mesenchyme, whilst expression of the pan-endothelial marker CD31 is completely lost. A subset of epicardial cells (which do not express ENG protein) delaminate and undergo a similar mesenchymal transition to form epicardially derived cells (EPDCs). This transient intra-myocardial mesenchymal cell population expresses low levels of ENG protein, similar to valve mesenchyme.In conclusion, ENG shows dynamic changes of expression in vascular endothelial cells, endocardial cells and mesenchymal cells in the developing heart that vary according to cardiovascular cell type.     

Protein expression profiles of Bos taurus blood and lymphatic vessel endothelial cells

The endothelium is a metabolically active organ that regulates the interaction between blood or lymph and the vessel or the surrounding tissue. Blood endothelium has been the object of many investigations whereas lymphatic endothelium biology is yet poorly understood. This report deals with a proteomic approach to the characterization and comparative analysis of lymphatic and blood vessel endothelial cells (ECs). By 2-DE we visualized the protein profiles of EC extracts from the thoracic aorta, inferior vena cava, and thoracic duct of Bos taurus. The three obtained electropherograms were then analyzed by specific software, and 113 quantitative and 25 qualitative differences were detected between the three endothelial gels. The cluster analysis of qualitative and quantitative differences evidenced the protein pattern of lymphatic ECs to be more similar to the venous than to the arterial one. Moreover, venous ECs were interestingly found showing a protein expression profile more similar to the lymphatic ECs than to the arterial ones. We also identified 64 protein spots by MALDI-TOF MS and ESI-IT MS/MS and three reference maps of bovine endothelium were obtained. The functional implications of the identified proteins in vascular endothelial biology are discussed.     

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.     

Endocardial Tip Cells in the Human Embryo – Facts and Hypotheses

Experimental studies regarding coronary embryogenesis suggest that the endocardium is a source of endothelial cells for the myocardial networks. As this was not previously documented in human embryos, we aimed to study whether or not endothelial tip cells could be correlated with endocardial-dependent mechanisms of sprouting angiogenesis. Six human embryos (43–56 days) were obtained and processed in accordance with ethical regulations; immunohistochemistry was performed for CD105 (endoglin), CD31, CD34, α-smooth muscle actin, desmin and vimentin antibodies. Primitive main vessels were found deriving from both the sinus venosus and aorta, and were sought to be the primordia of the venous and arterial ends of cardiac microcirculation. Subepicardial vessels were found branching into the outer ventricular myocardium, with a pattern of recruiting α-SMA+/desmin+ vascular smooth muscle cells and pericytes. Endothelial sprouts were guided by CD31+/CD34+/CD105+/vimentin+ endothelial tip cells. Within the inner myocardium, we found endothelial networks rooted from endocardium, guided by filopodia-projecting CD31+/CD34+/CD105+/ vimentin+ endocardial tip cells. The myocardial microcirculatory bed in the atria was mostly originated from endocardium, as well. Nevertheless, endocardial tip cells were also found in cardiac cushions, but they were not related to cushion endothelial networks. A general anatomical pattern of cardiac microvascular embryogenesis was thus hypothesized; the arterial and venous ends being linked, respectively, to the aorta and sinus venosus. Further elongation of the vessels may be related to the epicardium and subepicardial stroma and the intramyocardial network, depending on either endothelial and endocardial filopodia-guided tip cells in ventricles, or mostly on endocardium, in atria.     

Outflow pathways of venous blood from the myocardium in the dog and structures participating in their regulation

The intramural pathways of the venous blood outflow from the cardiac wall have been studied histologically, histochemically and micrometrically in 20 control and 84 experimental dogs with an artificially produced circulatory disturbances, peculiar for congenital heart disease (open arterial canal, coarctation of the aorta and stenosis of the pulmonary trunk). The experimental animals have been observed for 6-12 months. In the venous line of the coronary basin several morphologically differed parts, anatomically and functionally connected between themselves and ensuring blood outflow from the myocardium, are distinguished: coronary sinus, subepicardial veins, paired sinusoid veins, myocardial sinusoids and endocardial cushions. In each of them there are their own adaptive structures, participating in regulation of the venous blood stream. In the cardial sinus, in the subepicardial and paired sinusoid veins--these are valves of various complexity. In the myocardial sinusoids, the regulatory function, together with the valves, are performed by the intimal and muscle cushions, connective tissue and muscle bridges. In the endocardial cushions they are realized by the valves, muscle sphincters, bundles of obliquely and longitudinally oriented leiomyocytes. All the adaptive structures mentioned are also found in the hearts of the control animals. Under modelling various hemodynamic disturbances, the degree of their development increases sharply. The latter ensures the maintenance of an optimal regimen of blood circulation in the myocardium of a functionally loaded heart and prevents development of decompensation in the organ.     

Bmp2 instructs cardiac progenitors to form the heart-valve-inducing field

A hallmark of heart-valve development is the swelling and deposition of extracellular matrix in the heart-valve region. Only myocardium overlying this region can signal to underlying endothelium and cause it to lose cell-cell contacts, delaminate, and invade the extracellular space abutting myocardium and endocardium to form endocardial cushions (EC) in a process known as epithelial to mesenchymal transformation (EMT). The heart-valve myocardium expresses bone morphogenetic protein-2 (Bmp2) coincident with development of valve mesenchyme. BMPs belong to the transforming growth factor beta superfamily (TGF-beta) and play a wide variety of roles during development. We show that conditional ablation of Bmp2 in cardiac progenitors results in cell fate changes in which the heart-valve region adopts the identity of differentiated chamber myocardium. Moreover, Bmp2-deficient hearts fail to induce production and deposition of matrix at the heart-valve-forming region, resulting in the inability of the endothelium to swell and impairing the development of ECs. Furthermore, in collagen invasion assays, Bmp2 mutant endothelium is incapable of undergoing EMT, and addition of BMP2 protein to mutant heart explants rescues this phenotype. Our results demonstrate that Bmp2 is both necessary and sufficient to specify a field of cardiac progenitor cells as the heart-valve-inducing region amid developing atria and ventricles.     

Cellular localization of the endothelin receptor subtypes ET(A) and ET(B) in the rat heart and their differential expression in coronary arteries,veins, and capillaries

In the heart, the endothelin (ET)/endothelin-receptor system is markedly involved in pathophysiological mechanisms underlying various cardiac diseases. Based upon pharmacological studies both ET-receptor subtypes take part in the regulation of coronary vascular tone, however, their detailed cellular distribution in the coronary vascular bed based upon direct mRNA and protein detection is unknown. This issue was addressed in the rat heart by means of non-radioactive in situ hybridization, RT-PCR, and immunohistochemistry. Expression of vascular ET(A)-receptors was detected in arterial smooth muscle and capillary endothelium while ET(B)-receptors were present in arterial, venous, and capillary endothelium, and in arterial and venous smooth muscle cells. This differential distribution of the ET-receptor subtypes supports the concept that ET(A)- as well as ET(B)-receptors mediate arterial vasoconstriction, while postcapillary vascular resistance is exclusively regulated by ET(B)-receptors. The observed capillary endothelial expression of the ET(A)-receptor correlates with the known ability of ET(A)-receptor antagonists to attenuate increases in cardiac microvascular permeability during endotoxin shock and ischemia/reperfusion injury.     

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.     

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.     

The mysterious origins of coronary vessels

The origin of the coronary vessels remains a mystery. Here we discuss recent studies that address this puzzle, including new work by Tian et al. recently published in Cell Research.We face a growing epidemic of coronary vascular disease. Better understanding of the development of this unique vascular system will allow development of new treatment strategies. The origin of the coronary vessels has been a longstanding mystery. Classical anatomists proposed several potential sources for coronary vessels: the proepicardium (PE), the liver, the sinus venosus (SV) and the endocardium (Figure 1). Several recent reports have used sophisticated molecular and cell biological approaches to address this mystery, but have come to apparently contradictory conclusions. Tian et al.¹ use new lineage-tracing approaches to solve this puzzle, leading to new insights and new questions.Open in a separate windowFigure 1Diagram of E9.5 mouse embryo illustrating the proposed sources of coronary ECs. sv, sinus venosus; pe, proepicardium; li, liver primordium; v, ventricle; a, atrium.Initial studies in avian embryos, based on clonal retroviral labeling, dye labeling and quail-chick interspecies chimeras, indicated that coronary vascular smooth muscle and endothelial cells (vSMCs and ECs) derive from extracardiac sources. Most studies pinpointed the PE, a transient embryonic outgrowth of the septum transversum, as the cell source². PE cells transit to the heart, where they undergo an epithelial to mesenchymal transition (EMT). Based on these data, the predominant view from the early 1990s through the mid-2000s was that coronary vessels formed through a vasculogenic process from PE-derived mesenchymal cells. However, not all studies were in agreement. For example, Poelmann et al.³ reached a different conclusion and identified the nearby liver primoridium as the cell source. This study concluded that ECs and precursors formed small vessels that initially connected to the SV and then to subepicardial cells overlying the myocardium, which subsequently penetrated the myocardium to form the coronary vessels.The mainstream view of coronary artery formation from PE-derived ECs has been re-evaluated over the past decade through the use of Cre-LoxP genetic lineage-tracing approaches in mice^4,5,6,7. Several different mouse Cre lines that label populations within the PE were developed. Although these lines generally robustly label coronary vSMCs, they label a low fraction of coronary ECs (generally < 10%). Superficially, this suggests a divergence between avian and mammalian systems, but detailed comparison suggests that the results may be entirely consistent: the avian data indicate that some coronary ECs arise from the PE but the fraction of ECs that originate from PE was not determined. Both avian and murine studies could therefore be interpreted to suggest that a small fraction of coronary ECs arise from PE. A recent study further pointed out that PE contains heterogeneous cell populations, and some of these subpopulations (e.g., Sema3d⁺) contribute more robustly to coronary ECs than others (e.g., Tbx18⁺)⁷. Some lineages traced from the PE also contributed to ECs in the SV and endocardium, providing alternative routes whereby PE may give rise to coronary ECs. This study did not define the fraction of coronary ECs labeled by any of these subpopulations, therefore an estimate of the extent that these additional PE subpopulations contribute to coronary ECs is currently unavailable.Red-Horse et al.⁸ recently re-examined the endothelial lining of the SV as the origin of coronary ECs. Consistent with the study by Poelmann et al.³ in avian embryos, Red-Horse et al. observed that the first vessels of the heart tube connect to the SV. Elegant clonal labeling experiments using an EC-specific, tamoxifen-induced Cre (Cdh5-CreERT2) showed that labeling of single cells around E7.5 yielded descendant “clones” of ECs. At this point in development, PE cells do not express CDH5 and therefore these clones do not originate from this source. Most clones (74%) included SV ECs. However, its relationships with extracardiac structures, such as the liver primordium, were not investigated. Interestingly, SV ECs express venous markers, but descendant ECs belong to arterial and venous lineages. Based on these data, Red-Horse et al. concluded that most coronary ECs arise by angiogenic sprouting of SV ECs onto the developing heart, where they dedifferentiate, proliferate, form the coronary plexus, and subsequently redifferentiate into coronary arteries, capillaries and veins. While these data are compelling, to what extent this mechanism contributes to the coronary vasculature cannot be determined from this study.Wu et al.⁹ used a different lineage-tracing strategy to study coronary vessel origins and reached a different conclusion. This study was based on both constitutive and inducible Cre alleles driven by endocardium-specific Nfatc1 regulatory elements, which do not label PE, epicardium or SV prior to E10.5. By clonal analysis, Nfatc1-lineage cells differentiated to both artery and veins. Quantitative analysis showed that Nfatc1-labeled ECs form most intramyocardial coronary ECs (predominantly arteries) and a minority of supepicardial coronary ECs (predominantly veins). The clonal analysis of Red-Horse et al.⁸ also identified endocardial budding as a source of coronary vessels. Their data showed that fewer clones (24%) contained endocardial cells compared to SV cells, leading to the conclusion that endocardium makes a lesser contribution compared to the SV. However, this assumes equivalent labeling by Cdh5-CreERT2 under conditions where tamoxifen levels were limited. The frequency of endocardial cell labeling under these conditions may have been lower, for example if endocardial cells express lower levels of CreERT2.Tian et al.¹ studied coronary vessel development using Apln^CreERT2, a new lineage-tracing tool that selectively labels newly forming vessels but not established vessels or endocardium. Well-executed morphological and lineage-tracing experiments provide strong evidence that Apln^CreERT2 pulse activation at E11.5 labels nearly all subepicardial and intramyocardial coronary vessels of the ventricular free walls. Pulse labeling at this time labeled only rare ECs in the ventricular septum, suggesting that these vessels arise from ECs that express Apln^CreERT2 only after E11.5 and not from labeled ECs already present in the ventricular free walls. The endocardium appears to be an excellent candidate source for ECs in the ventricular septum. Clonal labeling experiments further demonstrated that at the single cell level, Apln⁺ ECs, named subepicardial ECs, retain the potential to differentiate into both arteries and veins.What is the relationship between subepicardial ECs and the proposed sites of origin for coronary ECs (PE, SV, endocardium, and liver primordium)? Using in vitro organ culture, Tian et al.¹ show that these cells are generated from the SV and subsequently extend onto the ventricles. Ventricles (containing ventricular endocardium) did not generate these cells in this system, leading the authors to conclude that they arise from the SV. However, the in vitro system does not yield robust coronary vessel formation, and it is entirely possible that certain developmental processes, such as endocardial budding or epicardial differentiation, are inactive under these conditions. Thus, we can conclude that some Apln⁺ ECs arise from SV, but the possibility of their origin also from other sources such as endocardium, PE, or liver primordium cannot be excluded.In summary, coronary ECs arise from multiple sources, and the balance between sources likely differs by anatomic region. While many studies on coronary vessel origins appear to reach conflicting conclusions, careful considerations of the experimental approaches and their limitations suggest models consistent with most published data. For instance, perhaps endocardial budding generates most intramyocardial coronaries, while angiogenic sprouting from the SV generates most subepicardial coronaries and a subset of intramyocardial coronaries. PE cells may contribute to a fraction of both EC populations, and give rise to most of the supporting smooth muscle cells. The Apln⁺ subepicardial ECs may represent a key common intermediate formed from all of these sources. Evaluating the contribution of each proposed cell source to this population will be important to understand the origins and growth patterns of coronary vessels. Further progress will depend on carefully quantitating the contribution of various EC sources to coronary vessel subtypes stratified by anatomic location.Understanding the origins of coronary vessels has implications for therapeutic strategies for coronary artery diseases, as each cell source suggests distinct mechanisms. For instance, SV angiogenic sprouting would direct us to investigate the signals that induce SV EC dedifferentiation and then redifferentiation into artery and vein ECs. PE-derived ECs might be induced by enhancing adult epicardial EMT and EC differentiation, while an endocardial EC source would prompt us to understand the signals that regulate the endocardial budding and differentiation process. The work of Tian et al. and the many other studies summarized herein are yielding insights into the mystery of coronary vessel origins. Solving this puzzle will yield rich rewards.     

Adaptive structures of the venous bed of the heart in normal state and in congenital defects

By means of a complex of anatomical, morphometrical and histological methods in 45 normally formed and in 179 abnormally developed hearts from persons of both sex died at various age, various links of the venous blood outflow from the myocardium have been investigated. Various parts, differing in a number of morphological signs, have been distinguished: coronary sinus, subepicardial veins, paired sinusoid veins, sinusoids of the myocardium and endocardial eversions. Regulation of their blood stream is performed by a system of simple muscular and infundibular valves, by intimal and muscular cushions, by connective tissue and muscular bridges. These adaptive structures occur in the normal heart also, but at congenital heart diseases, however, they reach an essentially greater development. Their localization is predominantly in the area of venular, vein, sinusoid bifurcations and endocardial eversions. Regular functioning of some parts of them is performed not by nonstriated, but by the cardiac muscular tissue. The coordinating work of the formation in question is one of the factors, that ensures the state of compensation in the defectively formed heart.     

Endothelial S1pr1 regulates pressure overload‐induced cardiac remodelling through AKT‐eNOS pathway

Cardiac vascular microenvironment is crucial for cardiac remodelling during the process of heart failure. Sphingosine 1‐phosphate (S1P) tightly regulates vascular homeostasis via its receptor, S1pr1. We therefore hypothesize that endothelial S1pr1 might be involved in pathological cardiac remodelling. In this study, heart failure was induced by transverse aortic constriction (TAC) operation. S1pr1 expression is significantly increased in microvascular endothelial cells (ECs) of post‐TAC hearts. Endothelial‐specific deletion of S1pr1 significantly aggravated cardiac dysfunction and deteriorated cardiac hypertrophy and fibrosis in myocardium. In vitro experiments demonstrated that S1P/S1pr1 praxis activated AKT/eNOS signalling pathway, leading to more production of nitric oxide (NO), which is an essential cardiac protective factor. Inhibition of AKT/eNOS pathway reversed the inhibitory effect of EC‐S1pr1‐overexpression on angiotensin II (AngII)‐induced cardiomyocyte (CM) hypertrophy, as well as on TGF‐β‐mediated cardiac fibroblast proliferation and transformation towards myofibroblasts. Finally, pharmacological activation of S1pr1 ameliorated TAC‐induced cardiac hypertrophy and fibrosis, leading to an improvement in cardiac function. Together, our results suggest that EC‐S1pr1 might prevent the development of pressure overload‐induced heart failure via AKT/eNOS pathway, and thus pharmacological activation of S1pr1 or EC‐targeting S1pr1‐AKT‐eNOS pathway could provide a future novel therapy to improve cardiac function during heart failure development.     

Histological and immunocytochemical investigation of human coronary vessel development with anti-CD34 antibodies

Information about embryonic development of coronary endothelium is the main clue for the creation of new methods in tissue engineering for treatment of ischemic heart diseases. The purpose of the research was to describe human coronary vessels development on early stages of the prenatal ontogenesis. The first step in human coronary vessels development is the formation of endothelium de novo by transformation of some epicardial and, possibly, endocardial cells. The next step is the ingrowth of sinus venosus endothelium in subepicardium over ventricles and atria, which gives rise to the coronary vessels. Only after 7 days does the primitive coronary plexus of the heart communicate with aorta (third step). During this period, some subepicardial vessels invade myocardium and some intramyocardial vessels contact with the heart cavity. Such intercommunications could help in regulation of blood circulation in primitive coronary plexus before establishment of effective contacts between arterial and venous vessels—excess of blood could be discharged directly into the heart cavity. Additional population of CD34+ cells were revealed inside condensed mesenchyme of the conotruncus; it participates in the formation of vasa vasorum in the aorta. Epicardium and sinus venosus generate endothelium of coronary vessels by neovasculo- and angiogenesis, respectively. During a week after ingrowth of vessels from SV and before their ingrowth to the aorta, ventriculo-coronary communications could be found in the heart.     

Pdgfrb‐Cre targets lymphatic endothelial cells of both venous and non‐venous origins

The Pdgfrb‐Cre line has been used as a tool to specifically target pericytes and vascular smooth muscle cells. Recent studies showed additional targeting of cardiac and mesenteric lymphatic endothelial cells (LECs) by the Pdgfrb‐Cre transgene. In the heart, this was suggested to provide evidence for a previously unknown nonvenous source of LECs originating from yolk sac (YS) hemogenic endothelium (HemEC). Here we show that Pdgfrb‐Cre does not, however, target YS HemEC or YS‐derived erythro‐myeloid progenitors (EMPs). Instead, a high proportion of ECs in embryonic blood vessels of multiple organs, as well as venous‐derived LECs were targeted. Assessment of temporal Cre activity using the R26‐mTmG double reporter suggested recent occurrence of Pdgfrb‐Cre recombination in both blood and lymphatic ECs. It thus cannot be excluded that Pdgfrb‐Cre mediated targeting of LECs is due to de novo expression of the Pdgfrb‐Cre transgene or their previously established venous endothelial origin. Importantly, Pdgfrb‐Cre targeting of LECs does not provide evidence for YS HemEC origin of the lymphatic vasculature. Our results highlight the need for careful interpretation of lineage tracing using constitutive Cre lines that cannot discriminate active from historical expression. The early vascular targeting by the Pdgfrb‐Cre also warrants consideration for its use in studies of mural cells. genesis 54:350–358, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.     

Simultaneous isolation of endothelial and smooth muscle cells from human umbilical artery or vein and their growth response to low-density lipoproteins

In the pathogenesis of atherosclerosis the interplay of endothelial cells (ECs) and smooth muscle cells (SMCs) is disturbed. Oxidatively modified low-density lipoproteins (oxLDLs), important stimulators of atherosclerotic plaque formation in vessels, modify the growth response of both cell types. To compare growth responses of ECs and SMCs of the same vessel with oxLDLs, we developed a method to isolate both cell types from the vessel walls of umbilical cords by enzymatic digestion. The method further allowed the simultaneous isolation of venous and arterial cells from a single umbilical cord. In culture, venous ECs showed an elongated appearance compared with arterial ECs, whereas SMCs of artery and vein did not look different. Smooth muscle cells of both vessel types responded to oxLDLs (60 microg/ml) with an increase in their [(3)H]-thymidine incorporation into DNA. On the contrary, ECs of artery or vein decreased [(3)H]-thymidine incorporation and cell number in the presence of oxLDLs (60 microg/ml) of increasing oxidation grade. Thus, human umbilical SMCs and ECs of the same vessel show a disparate growth response toward oxLDLs. But the physiologically more relevant minimal oxLDLs did not decrease proliferation in venous ECs but only in arterial ECs. This difference in tolerance toward minimal oxLDLs should be taken into account while using venous or arterial ECs of umbilical cord for research in atherosclerosis. Further differences of venous and arterial ECs in tolerance toward minimal oxLDLs could be of clinical relevance for coronary artery bypass grafts.