Sphingolipids and the orchestration of endothelium-derived vasoactive factors: When endothelial function demands greasing

Vasomotor tone is regulated by a complex interplay of a variety of extrinsic neurohumoral and intrinsic factors. It is the endothelium that has a major influence on smooth muscle cell tone via the release of intrinsic vasoactive factors and is therefore an important regulator of vasomotor tone. Sphingolipids are an emerging class of lipid mediators with important physiological properties. In the last two decades it has not only become increasingly clear that sphingolipid signaling plays a pivotal role in immune function, but also its role in the vascular system is now becoming more recognized. In this mini-review we will highlight the possible cross-talk between sphingolipids and intrinsic vasoactive factors released by the endothelium. Via this cross-talk sphingolipids can orchestrate vasomotor tone and may therefore also be involved in the pathophysiology of disease states associated with endothelial dysfunction.     

Assembly of trunk and limb blood vessels involves extensive migration and vasculogenesis of somite-derived angioblasts

Vascular development requires the assembly of precursor cells into blood vessels, but how embryonic vessels are assembled is not well understood. To determine how vascular cells migrate and assemble into vessels of the trunk and limb, marked somite-derived angioblasts were followed in developing embryos. Injection of avian somites with the cell-tracker DiI showed that somite-derived angioblasts in unperturbed embryos migrated extensively and contributed to trunk and limb vessels. Mouse-avian chimeras with mouse presomitic mesoderm grafts had graft-derived endothelial cells in blood vessels at significant distances from the graft, indicating that mouse angioblasts migrated extensively in avian hosts. Mouse graft-derived endothelial cells were consistently found in trunk vessels, such as the perineural vascular plexus, the cardinal vein, and presumptive intersomitic vessels, as well as in vessels of the limb and kidney rudiment. This reproducible pattern of graft colonization suggests that avian vascular patterning cues for trunk and limb vessels are recognized by mammalian somitic angioblasts. Mouse-quail chimeras stained with both the quail vascular marker QH1 and the mouse vascular marker PECAM-1 had finely chimeric vessels, with graft-derived mouse cells interdigitated with quail vascular cells in most vascular beds colonized by graft cells. Thus, diverse trunk and limb blood vessels have endothelial cells that developed from migratory somitic angioblasts, and assembly of these vessels is likely to have a large vasculogenic component.     

Origin of coronary endothelial cells from epicardial mesothelium in avian embryos

It has been established that coronary vessels develop through self-assembly of mesenchymal vascular progenitors in the subepicardium. Mesenchymal precursors of vascular smooth muscle cells and fibroblasts are known to originate from an epithelial-to-mesenchymal transformation of the epicardial mesothelium, but the origin of the coronary endothelium is still obscure. We herein report that at least part of the population of the precursors of the coronary endothelium are epicardially-derived cells (EPDCs). We have performed an EPDC lineage study through retroviral and fluorescent labelling of the proepicardial and epicardial mesothelium of avian embryos. In all the experiments onlythe surface mesothelium was labelled after 3 h of reincubation. However, endothelial cells from subepicardial vessels were labelled after 24-48 h and endothelial cells of intramyocardial vessels were also labelled after 48-96 h of reincubation. In addition, the development of the coronary vessels was studied in quail-chick chimeras, obtaining results which also support a mesothelial origin for endothelial and smooth muscle cells. Finally, quail proepicardial explants cultured on Matrigel showed colocalization of cytokeratin and QH1 (mesothelial and endothelial markers, respectively) after 24 h. These results, taken together, suggest that EPDC show similar competence to that displayed by bipotential vascular progenitor cells [Yamashita et al., Nature 408: 92-96 (2000)] which are able to differentiate into endothelium or smooth muscle depending on their exposure to VEGF or PDGF-BB. It is conceivable that the earliest EPDC differentiate into endothelial cells in response to myocardially-secreted VEGF, while further EPDC would be recruited by the nascent capillaries via PDGFR-beta signalling, giving rise to mural cells.     

Fibroblast growth factor 9 delivery during angiogenesis produces durable, vasoresponsive microvessels wrapped by smooth muscle cells

The therapeutic potential of angiogenic growth factors has not been realized. This may be because formation of endothelial sprouts is not followed by their muscularization into vasoreactive arteries. Using microarray expression analysis, we discovered that fibroblast growth factor 9 (FGF9) was highly upregulated as human vascular smooth muscle cells (SMCs) assemble into layered cords. FGF9 was not angiogenic when mixed with tissue implants or delivered to the ischemic mouse hind limb, but instead orchestrated wrapping of SMCs around neovessels. SMC wrapping in implants was driven by sonic hedgehog-mediated upregulation of PDGFRβ. Computed tomography microangiography and intravital microscopy revealed that microvessels formed in the presence of FGF9 had enhanced capacity to receive flow and were vasoreactive. Moreover, the vessels persisted beyond 1 year, remodeling into multilayered arteries paired with peripheral nerves. This mature physiological competency was attained by targeting mesenchymal cells rather than endothelial cells, a finding that could inform strategies for therapeutic angiogenesis and tissue engineering.     

Human fetal aorta-derived vascular progenitor cells: identification and potential application in ischemic diseases

Vasculogenesis, the formation of blood vessels in embryonic or fetal tissue mediated by immature vascular cells (i.e., angioblasts), is poorly understood. Here we report a summary of our recent studies on the identification of a population of vascular progenitor cells (VPCs) in human fetal aorta. These undifferentiated mesenchymal cells co-express endothelial and myogenic markers (CD133+, CD34+, KDR+, desmin+) and are localized in outer layer of the aortic stroma of 11–12 weeks old human fetuses. Under stimulation with VEGF-A or PDGF-BB, VPCs give origin to a mixed population of mature endothelial and mural cells, respectively. When embedded in a three-dimensional collagen gel, VPCs organize into cohesive cellular cords that resembled mature vascular structures. The therapeutic efficacy of a small number of VPCs transplanted into ischemic limb muscle was demonstrated in immunodeficient mice. Investigation of the effect of VPCs on experimental heart ischemia and on diabetic ischemic ulcers in mice is in progress and seems to confirm their efficacy. On the whole, fetal aorta represents an important source for the investigation of phenotypic and functional features of human vascular progenitor cells.     

Loss of nidogen-1 and -2 results in syndactyly and changes in limb development

Nidogens are two ubiquitous basement membrane proteins produced mainly by mesenchymal cells. Nidogen-mediated interactions, in particular with laminin, collagen IV, and perlecan have been considered important in the formation and maintenance of the basement membrane. However, whereas mice lacking both nidogen isoforms or carrying mutations in the high affinity nidogen-binding site upon the laminin gamma1 chain have specific basement membrane defects in certain organs, particularly in the lung, characterization of these mice has also shown that basement membrane formation per se does not need nidogens or the laminin-nidogen interaction. Limb development requires the complex interplay of numerous growth factors whose expression is dependent upon the apical ectodermal ridge. Here, we show that lack of nidogen-1 and -2 results in a specific and time-limited failure in the ectodermal basement membrane of the limb bud. The absence of this basement membrane leads to aberrant apical ectodermal ridge formation. It also causes altered distribution of growth factors, such as fibroblast growth factors and leads to a fully penetrant soft tissue syndactyly caused by the dysregulation of interdigital apoptosis. Further, in certain animals more severe changes in bone formation occur, providing evidence for the interplay between growth factors and the extracellular matrix.     

Laser-guided direct writing for three-dimensional tissue engineering

One of the principal limitations to the size of an engineered tissue is oxygen and nutrient transport. Lacking a vascular bed, cells embedded in an engineered tissue will consume all available oxygen within hours while out branching blood vessels will take days to vascularize the implanted tissue. One possible solution is to directly write vascular structures within the engineered tissue prior to implantation, reconstructing the tissue according to its native architecture. The cell patterning technique, laser-guided direct writing (LGDW), can pattern multiple cells types with micrometer resolution on arbitrary surfaces, including biological gels. Here we show that LGDW can pattern human umbilical vein endothelial cells (HUVEC) in two- and three-dimensions with micrometer accuracy. By patterning HUVEC on Matrigel, we can direct their self-assembly into vascular structures along the desired pattern. Finally, co-culturing the vascular structures with hepatocytes resulted in an aggregated tubular structure similar in organization to a hepatic sinusoid. This capability can facilitate studies of tissue architecture at the single cell level, and of heterotypic interactions underlying processes such as liver and pancreas morphogenesis, differentiation, and angiogenesis.     

Development of avascularity during cartilage differentiation in the embryonic limb

The differentiation of cartilage and muscle in limb-bud mesenchyme has been interpreted by some investigators in terms of a vascular pre-pattern model. It has been argued that a pre-pattern of the early limb vasculature compartmentalises the mesenchyme into specific microenvironmental areas in which, depending on the oxygen tension and nutrient supply, cartilage or muscle will differentiate. However, recent analyses of the development and differentiation of blood vessels in limbs have shown that regional variations in vascularization develop co-incidentally with the earliest indication of cartilage formation or mesenchymal condensation. The simple model described in the present study suggests that the mechanical compression/tension forces generated by the condensing mesenchyme are sufficient to constrict and eventually close off the thin-walled undifferentiated vessels caught in the condensation foci, thus leading to the avascularity of cartilage rudiments. This view suggests that the vasculature has no major function in governing the pattern of cartilage differentiation.     

Ephrin-A2 regulates position-specific cell affinity and is involved in cartilage morphogenesis in the chick limb bud

In the developing limb bud, mesenchymal cells show position-specific affinity, suggesting that the positional identity of the cells is represented as their surface properties. Since the affinity is regulated by glycosylphosphatidylinositol (GPI)-anchored cell surface proteins, and by EphA4 receptor tyrosine kinase, we hypothesized that the GPI-anchored ligand, the ephrin-A family, also contributes to the affinity. Here, we describe the role of ephrin-A2 in the chick limb bud. Ephrin-A2 protein is uniformly distributed in the limb bud during early limb development. As the limb bud grows, expression of ephrin-A2 is strong in its proximal-to-intermediate regions, but weak distally. The position-dependent expression is maintained in vitro, and is regulated by FGF protein, which is produced in the apical ectodermal ridge. To investigate the role of ephrin-A2 in affinity and in cartilage morphogenesis of limb mesenchyme, we ectopically expressed ephrin-A2 in the limb bud using the retrovirus vector, RCAS. Overexpressed ephrin-A2 modulated the affinity of the mesenchymal cells that differentiate into autopod elements. It also caused malformation of the autopod skeleton and interfered with cartilage nodule formation in vitro without inhibiting chondrogenesis. These results suggest that ephrin-A2 regulates the position-specific affinity of limb mesenchyme and is involved in cartilage pattern formation in the limb.     

Dissecting coronary angiogenesis: 3D co-culture of cardiomyocytes with endothelial or mesenchymal cells

In embryogenesis, coronary blood vessels are formed by vasculogenesis from epicardium-derived progenitors. Subsequently, growing or regenerating myocardium increases its vasculature by angiogenesis, forming new vessels from the pre-existing ones. Recently, cell therapies for myocardium ischemia that used different protocols have given promising results, using either extra-cardiac blood vessel cell progenitors or stimulating the cardiac angiogenesis. We have questioned whether cardiomyocytes could sustain both vasculogenesis and angiogenesis. We used a 3D culture model of tissue-like spheroids in co-cultures of cardiomyocytes supplemented either with endothelial cells or with bone marrow-derived mesenchymal stroma cells. Murine foetal cardiomyocytes introduced into non-adherent U-wells formed 3D contractile structures. They were coupled by gap junctions. Cardiomyocytes segregated inside the 3D structure into clumps separated by connective tissue septa, rich in fibronectin. Three vascular endothelial growth factor isoforms were produced (VEGF 120, 164 and 188). When co-cultured with human umbilical cord endothelial cells, vascular structures were produced in fibronectin-rich external layer and in radial septa, followed by angiogenic sprouting into the cardiomyocyte microtissue. Presence of vascular structures led to the maintenance of long-term survival and contractile capacity of cardiac microtissues. Conversely, bone marrow mesenchymal cells formed isolated cell aggregates, which progressively expressed the endothelial markers von Willebrand's antigen and CD31. They proceeded to typical vasculogenesis forming new blood vessels organised in radial pattern. Our results indicate that the in vitro 3D model of cardiomyocyte spheroids provides the two basic elements for formation of new blood vessels: fibronectin and VEGF. Within the myocardial environment, endothelial and mesenchymal cells can proceed to formation of new blood vessels either through angiogenesis or vasculogenesis, respectively.     

Intrinsic versus extrinsic influences in the development of neuronal maps

Accumulating evidence suggests that the plasticity of extrinsic thalamocortical inputs in cortical layer IV may be guided or instructed by earlier plasticity events in the intrinsic, horizontal connections within the extragranular cortical layers. We analyse a rate-based model of the plasticity of a set of extrinsic afferents in the presence of a pre-existing (and fixed) plexus of intrinsic, overall excitatory horizontal connections between a set of target neurons. We determine conditions under which afferent synaptic pattern formation respects this pre-existing lateral structure. We find three broad regimes under which extrinsic afferent plasticity may violate this structure: the initial pattern of extrinsic afferent innervation of the target cells is far from balanced; the gain of the extrinsic afferents greatly exceeds the overall scale of the strength of lateral excitation; the target cell horizontal coupling matrix is sparse. If none of these conditions is satisfied, then extrinsic afferent plasticity respects the pre-existing lateral connectivity, so that afferent synaptic pattern formation conforms to the pattern of lateral excitation.     

When do mixotrophs specialize? Adaptive dynamics theory applied to a dynamic energy budget model

In evolutionary history, several events have occurred at which mixotrophs specialized into pure autotrophs and heterotrophs. We studied the conditions under which such events take place, using the Dynamic Energy Budget (DEB) theory for physiological rules of the organisms' metabolism and Adaptive Dynamics (AD) theory for evolutionary behavior of parameter values. We modeled a population of mixotrophs that can take up dissolved inorganic nutrients by autotrophic assimilation and detritus by heterotrophic assimilation. The organisms have a certain affinity for both pathways; mutations that occur in the affinities enable the population to evolve. One of the possible evolutionary outcomes is a branching point which provides an opportunity for the mixotrophic population to split up and specialize into separate autotrophs and heterotrophs. Evolutionary branching is not a common feature of the studied system, but is found to occur only under specific conditions. These conditions depend on intrinsic properties such as the cost function, the level of the costs and the boundaries of the trait space: only at intermediate cost levels and when an explicit advantage exists to pure strategies over mixed ones may evolutionary branching occur. Usually, such an advantage (and hence evolutionary branching) can be induced by interference between the two affinities, but this result changes due to the constraints on the affinities. Now, only some of the more complicated cost functions give rise to a branching point. In contrast to the intrinsic properties, extrinsic properties such as the total nutrient content or light intensity were found to have no effect on the evolutionary outcomes at all.     

Isolating and defining cells to engineer human blood vessels

A great deal of attention has been recently focused on understanding the role that bone marrow-derived putative endothelial progenitor cells (EPC) may play in the process of neoangiogenesis. However, recent data indicate that many of the putative EPC populations are comprised of various haematopoietic cell subsets with proangiogenic activity, but these marrow-derived putative EPC fail to display vasculogenic activity. Rather, this property is reserved for a rare population of circulating viable endothelial cells with colony-forming cell (ECFC) ability. Indeed, human ECFC possess clonal proliferative potential, display endothelial and not haematopoietic cell surface antigens, and display in vivo vasculogenic activity when suspended in an extracellular matrix and implanted into immunodeficient mice. Furthermore, human vessels derived became integrated into the murine circulatory system and eventually were remodelled into arterial and venous vessels. Identification of this population now permits determination of optimal type I collagen matrix microenvironment into which the cells should be embedded and delivered to accelerate and even pattern number and size of blood vessels formed, in vivo. Indeed, altering physical properties of ECFC-collagen matrix implants changed numerous parameters of human blood vessel formation, in host mice. These recent discoveries may permit a strategy for patterning vascular beds for eventual tissue and organ regeneration.     

骨与关节系统发生过程中相关调控机制的研究进展

脊椎动物胚胎期骨与关节系统的发生是一种复杂生命现象,起始于中胚层间充质细胞的定向聚集,形成肢芽,然后在一系列作用因子的调控下,肢芽内细胞进一步分化,形成具有骨骼雏形的软骨原基,后者经软骨内骨化发育成骨。四肢骨大多是以这种方式发生的,四肢的滑膜关节系统也随骨骼的发生而形成。详细阐述了近年来对肢体骨与关节系统发生各步骤相关调控机制方面的研究进展。Abstract: The embryonic development of bone and joint involves in complicated events for vertebrate limb. It originates from determined condensation of mesenchymal cells from lateral mesoderm. These cells and the overlying ectodermal jacket form limb buds at presumptive limb levels. Then, under the control of systemic factors, mesenchymal cells aggregate and differentiate to form catilage blastemal elements that prefigure skeletal limb components. The latter develops into skeleton through endochondral ossification. The majority of the bones of the limb form by the endochondral mechanism. The formation of synovial joint system and bone development occur simultaneously. This article reviewed the progress on the related control mechanism in the development of bone and joint recently.     

Origination and innovation in the vertebrate limb skeleton: an epigenetic perspective

The vertebrate limb has provided evolutionary and developmental biologists with grist for theory and experiment for at least a century. Its most salient features are its pattern of discrete skeletal elements, the general proximodistal increase in element number as development proceeds, and the individualization of size and shape of the elements in line with functional requirements. Despite increased knowledge of molecular changes during limb development, however, the mechanisms for origination and innovation of the vertebrate limb pattern are still uncertain. We suggest that the bauplan of the limb is based on an interplay of genetic and epigenetic processes; in particular, the self-organizing properties of precartilage mesenchymal tissue are proposed to provide the basis for its ability to generate regularly spaced nodules and rods of cartilage. We provide an experimentally based "core" set of cellular and molecular processes in limb mesenchyme that, under realistic conditions, exhibit the requisite self-organizing behavior for pattern origination. We describe simulations that show that under limb bud-like geometries the core mechanism gives rise to skeletons with authentic proximodistal spatiotemporal organization. Finally, we propose that evolution refines skeletal templates generated by this process by mobilizing accessory molecular and biomechanical regulatory processes to shape the developing limb and its individual elements. Morphological innovation may take place when such modulatory processes exceed a threshold defined by the dynamics of the skeletogenic system and elements are added or lost.     

Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction

Mesenchymal stem cells are multipotent cells that can differentiate into cardiomyocytes and vascular endothelial cells. Here we show, using cell sheet technology, that monolayered mesenchymal stem cells have multipotent and self-propagating properties after transplantation into infarcted rat hearts. We cultured adipose tissue-derived mesenchymal stem cells characterized by flow cytometry using temperature-responsive culture dishes. Four weeks after coronary ligation, we transplanted the monolayered mesenchymal stem cells onto the scarred myocardium. After transplantation, the engrafted sheet gradually grew to form a thick stratum that included newly formed vessels, undifferentiated cells and few cardiomyocytes. The mesenchymal stem cell sheet also acted through paracrine pathways to trigger angiogenesis. Unlike a fibroblast cell sheet, the monolayered mesenchymal stem cells reversed wall thinning in the scar area and improved cardiac function in rats with myocardial infarction. Thus, transplantation of monolayered mesenchymal stem cells may be a new therapeutic strategy for cardiac tissue regeneration.     

Involvement of vessels and PDGFB in muscle splitting during chick limb development

Muscle formation and vascular assembly during embryonic development are usually considered separately. In this paper, we investigate the relationship between the vasculature and muscles during limb bud development. We show that endothelial cells are detected in limb regions before muscle cells and can organize themselves in space in the absence of muscles. In chick limbs, endothelial cells are detected in the future zones of muscle cleavage, delineating the cleavage pattern of muscle masses. We therefore perturbed vascular assembly in chick limbs by overexpressing VEGFA and demonstrated that ectopic blood vessels inhibit muscle formation, while promoting connective tissue. Conversely, local inhibition of vessel formation using a soluble form of VEGFR1 leads to muscle fusion. The endogenous location of endothelial cells in the future muscle cleavage zones and the inverse correlation between blood vessels and muscle suggests that vessels are involved in the muscle splitting process. We also identify the secreted factor PDGFB (expressed in endothelial cells) as a putative molecular candidate mediating the muscle-inhibiting and connective tissue-promoting functions of blood vessels. Finally, we propose that PDGFB promotes the production of extracellular matrix and attracts connective tissue cells to the future splitting site, allowing separation of the muscle masses during the splitting process.