Notch signaling is required for arterial-venous differentiation during embryonic vascular development

Recent evidence indicates that acquisition of artery or vein identity during vascular development is governed, in part, by genetic mechanisms. The artery-specific expression of a number of Notch signaling genes in mouse and zebrafish suggests that this pathway may play a role in arterial-venous cell fate determination during vascular development. We show that loss of Notch signaling in zebrafish embryos leads to molecular defects in arterial-venous differentiation, including loss of artery-specific markers and ectopic expression of venous markers within the dorsal aorta. Conversely, we find that ectopic activation of Notch signaling leads to repression of venous cell fate. Finally, embryos lacking Notch function exhibit defects in blood vessel formation similar to those associated with improper arterial-venous specification. Our results suggest that Notch signaling is required for the proper development of arterial and venous blood vessels, and that a major role of Notch signaling in blood vessels is to repress venous differentiation within developing arteries. Movies available on-line     

Plasticity of endothelial cells during arterial-venous differentiation in the avian embryo

Remodeling of the primary vascular system of the embryo into arteries and veins has long been thought to depend largely on the influence of hemodynamic forces. This view was recently challenged by the discovery of several molecules specifically expressed by arterial or venous endothelial cells. We here analysed the expression of neuropilin-1 and TIE2, two transmembrane receptors known to play a role in vascular development. In birds, neuropilin-1 was expressed by arterial endothelium and wall cells, but absent from veins. TIE2 was strongly expressed in embryonic veins, but only weakly transcribed in most arteries. To examine whether endothelial cells are committed to an arterial or venous fate once they express these specific receptors, we constructed quail-chick chimeras. The dorsal aorta, carotid artery and the cardinal and jugular veins were isolated together with the vessel wall from quail embryos between embryonic day 2 to 15 and grafted into the coelom of chick hosts. Until embryonic day 7, all grafts yielded endothelial cells that colonized both host arteries and veins. After embryonic day 7, endothelial plasticity was progressively lost and from embryonic day 11 grafts of arteries yielded endothelial cells that colonized only chick arteries and rarely reached the host veins, while grafts of jugular veins colonized mainly host veins. When isolated from the vessel wall, quail aortic endothelial cells from embryonic day 11 embryos were able to colonize both host arteries and veins. Our results show that despite the expression of arterial or venous markers the endothelium remains plastic with regard to arterial-venous differentiation until late in embryonic development and point to a role for the vessel wall in endothelial plasticity and vessel identity.     

Club endings containing substance P-like immunoreactivity in human retinal vessels: Whole mount preparation

Substance P-like immunoreactivity was studied by fluorescence immunohistochemistry in human retinal vessels of the whole mount preparation. Substance P immunoreactive nerve fibers were seen to run along the arteries and some veins. Club terminals are in contact with the vessel wall. Substance P may act as a vasoregulator in the human retina.     

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.     

Blood vessel buckling within soft surrounding tissue generates tortuosity

The stability of blood vessel under lumen pressure load is essential to the maintenance of normal arterial function. Previous mechanical models showed that blood vessels may buckle into a half sine wave but arteries and veins in vivo often demonstrate tortuous paths with multiple waves. The objective of this study was to analyze the buckling of blood vessels under lumen pressure with surrounding tissue support. Blood vessels were modeled as elastic cylindrical vessels within an elastic substrate. Buckling equations were established to determine the critical pressure and the wavelength. These equations and simulation results demonstrated that blood vessels do take higher order mode shapes when buckling inside an elastic substrate while they take the basal mode shape without the substrate. The wave number increases i.e. blood vessels take a higher mode shape, as the stiffness of the substrate increases. These results suggest that mechanical buckling is a possible mechanism for the development of tortuous blood vessels. The current model provides a powerful tool for further studying the tortuosity of arteries and veins.     

reg6 is required for branching morphogenesis during blood vessel regeneration in zebrafish caudal fins

Postnatal neovascularization is essential for wound healing, cancer progression, and many other physiological functions. However, its genetic mechanism is largely unknown. In this report, we study neovascularization in regenerating adult zebrafish fins using transgenic fish that express EGFP in blood vessel endothelial cells. We first describe the morphogenesis of regenerating vessels in wild-type animals and then the phenotypic analysis of a genetic mutation that disrupts blood vessel regeneration. In wild-type zebrafish caudal fins, amputated blood vessels heal their ends by 24 h postamputation (hpa) and then reconnect arteries and veins via anastomosis, to resume blood flow at wound sites by 48 hpa. The truncated vessels regenerate by first growing excess vessels to form unstructured plexuses, resembling the primary capillary plexuses formed during embryonic vasculogenesis. Interestingly, this mode of vessel growth switches by 8 days postamputation (dpa) to growth without a plexus intermediate. During blood vessel regeneration, vessel remodeling begins during early plexus formation and continues until the original vasculature pattern is reestablished at approximately 35 dpa. Temperature-sensitive mutants for reg6 have profound defects in blood vessel regeneration. At the restrictive temperature, reg6 regenerating blood vessels first fail to make reconnections between severed arteries and veins, and then form enlarged vascular sinuses rather than branched vascular plexuses. Reciprocal temperature-shift experiments show that reg6 function is required throughout plexus formation, but not during later growth. Our results suggest that the reg6 mutation causes defects in branch formation and/or angiogenic sprouting.     

A mathematical model of diffusional shunting of oxygen from arteries to veins in the kidney

To understand how arterial-to-venous (AV) oxygen shunting influences kidney oxygenation, a mathematical model of oxygen transport in the renal cortex was created. The model consists of a multiscale hierarchy of 11 countercurrent systems representing the various branch levels of the cortical vasculature. At each level, equations describing the reactive-advection-diffusion of oxygen are solved. Factors critical in renal oxygen transport incorporated into the model include the parallel geometry of arteries and veins and their respective sizes, variation in blood velocity in each vessel, oxygen transport (along the vessels, between the vessels and between vessel and parenchyma), nonlinear binding of oxygen to hemoglobin, and the consumption of oxygen by renal tissue. The model is calibrated using published measurements of cortical vascular geometry and microvascular Po(2). The model predicts that AV oxygen shunting is quantitatively significant and estimates how much kidney Vo(2) must change, in the face of altered renal blood flow, to maintain cortical tissue Po(2) at a stable level. It is demonstrated that oxygen shunting increases as renal Vo(2) or arterial Po(2) increases. Oxygen shunting also increases as renal blood flow is reduced within the physiological range or during mild hemodilution. In severe ischemia or anemia, or when kidney Vo(2) increases, AV oxygen shunting in proximal vascular elements may reduce the oxygen content of blood destined for the medullary circulation, thereby exacerbating the development of tissue hypoxia. That is, cortical ischemia could cause medullary hypoxia even when medullary perfusion is maintained. Cortical AV oxygen shunting limits the change in oxygen delivery to cortical tissue and stabilizes tissue Po(2) when arterial Po(2) changes, but renders the cortex and perhaps also the medulla susceptible to hypoxia when oxygen delivery falls or consumption increases.     

Arterial versus venous endothelial cells

Vascular endothelial cells (ECs) form the inner lining of all blood vessels from the largest artery and veins, viz., the aorta and venae cavae, respectively, to the capillaries that connect the arterial and venous systems. Because these two major conducting systems of the cardiovasculature differ functionally, it is not surprising that the physical makeup of arteries and veins, including the ECs that line their lumina, are also distinct. Although few would argue that the local environment contributes to the differences between arteries and veins, recent evidence has shown that the specification of arterial and venous identity is largely genetically determined. The authors are supported by NIH EY05318 and EY015435 (P.A.D.). Dr. dela Paz is supported by NRSA Institutional Research Training Grant T32 HL076115. Dr. D’Amore is a Research to Prevent Blindness Senior Scientific Investigator.     

Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin

Nerves and blood vessels are branched structures, but whether their branching patterns are established independently or coordinately is not clear. Here we show that arteries, but not veins, are specifically aligned with peripheral nerves in embryonic mouse limb skin. Mutations that eliminate peripheral sensory nerves or Schwann cells prevent proper arteriogenesis, while those that disorganize the nerves maintain the alignment of arteries with misrouted axons. In vitro, sensory neurons or Schwann cells can induce arterial marker expression in isolated embryonic endothelial cells, and VEGF(164/120) is necessary and sufficient to mediate this induction. These data suggest that peripheral nerves provide a template that determines the organotypic pattern of blood vessel branching and arterial differentiation in the skin, via local secretion of VEGF.     

Visualization of Intrapulmonary Lymph Vessels in Healthy and Inflamed Murine Lung Using CD90/Thy-1 as a Marker

Background

Lymphatic vessels play a pivotal role in fluid drainage and egress of immune cells from the lung. However, examining murine lung lymphatics is hampered by the expression of classical lymph endothelial markers on other cell types, which hinders the unambiguous identification of lymphatics. The expression of CD90/Thy-1 on lymph endothelium was recently described and we therefore examined its suitability to identify murine pulmonary lymph vessels under healthy and inflammatory conditions.

Methodology/Principal Findings

Immunohistochemistry with a monoclonal antibody against CD90.2/Thy-1.2 on 200 µm thick precision cut lung slices labeled a vascular network that was distinct from blood vessels. Preembedding immunostaining and electron microscopy verified that the anti-CD90.2/Thy-1.2 antibody labeled lymphatic endothelium. Absence of staining in CD90.1/Thy-1.1 expressing FVB mice indicated that CD90/Thy-1 was expressed on lymph endothelium and labeling was not due to antibody cross reactivity. Double-labeling immunohistochemistry for CD90/Thy-1 and α-smooth muscle actin identified two routes for lymph vessel exit from the murine lung. One started in the parenchyma or around veins and left via venous blood vessels. The other began in the space around airways or in the space between airways and pulmonary arteries and left via the main bronchi. As expected from the pulmonary distribution of lymph vessels, intranasal application of house dust mite led to accumulation of T cells around veins and in the connective tissue between airways and pulmonary arteries. Surprisingly, increased numbers of T cells were also detected around intraacinar arteries that lack lymph vessels. This arterial T cell sheath extended to the pulmonary arteries where lymph vessels were located.

Conclusions/Significance

These results indicate that CD90/Thy-1 is expressed on lymphatic endothelial cells and represents a suitable marker for murine lung lymph vessels. Combining CD90/Thy-1 labeling with precision cut lung slices allows visualizing the anatomy of the lymphatic system in normal and inflamed conditions.     

Study of the innervation of blood vessels of the digestive organs by fluorescence microscopy]

The blood vessels of the samll intestine and the gallbladder were shown to possess a great amount of adrenergic nerve fibres which, when penetrating the thickness of the wall of the above organs, become thinner and the distributed between the tissue structures of the organs as the thinnest monoaxonal network. The method of Falck--Hillarp--Krokhina was used. Among the vessel nerves there are perivascular nerves accompanying the vessels along their total legnth, juxtavascular and intramural nervous bundles of the sumpathetic nature detected by the fluorescent-microscopy method. Large arteries are disposed in a considerably thicker network of specifically fluorescing fibres than veins and small arteries.     

豚鼠小肠粘膜下层内神经节丛与血管的伴行现象

本文应用NADH黄递酶联合Karnovsky-Roots乙酰胆碱酯酶组化技术研究了豚鼠小肠粘膜下层铺片上神经节丛与血管的关系,结果发现,由肌层穿入粘膜下层的小动、静脉及其分支互相伴行,环绕肠壁;动、静脉两侧有大致平行走向的伴行神经节丛,并从伴行的神经节丛发出分支终止于动、静脉壁上以及连接动、静脉两侧的神经节丛之间有纤维束相连。这种伴行现象在小动、静脉起始段和第一级分支段最为明显。伴行的神经节多数呈长梭形,其长轴与血管长轴平行。在血管“人”字形分支处,可见到“人”字形神经节,常位于静脉叉内。上述结果提示,伴行的神经节丛可能调节粘膜下层和粘膜的血流,从而影响小肠的吸收功能。     

Parathyroid hormone-related protein-mediated responses in pulmonary arteries and veins of newborn lambs

PTHrP has important roles in lung development and function. Here we determined the vasomotor responses of isolated pulmonary arteries and veins of newborn and adult sheep to PTHrP. In vessels constricted with endothelin-1, PTHrP (PTHrP 1-34) caused greater relaxation of veins than of arteries. In both vessel types, relaxation to the peptide was less in adult than in newborn vessels. In newborn lambs, PTHrP-induced relaxation was not affected by endothelium removal, inhibition of eNOS, or inhibition of adenylyl cyclases by SQ-22536. However, relaxation was attenuated by 4-aminopyridine, inhibitor of voltage-dependent potassium channels, in both arteries and veins, and by charybdotoxin, inhibitor of calcium-activated potassium channels, in veins. When vessels were saturated with 8-BrcAMP (3 x 10(-4) M), to eliminate relaxation mediated by endogenous cAMP, PTHrP-induced relaxation was partially attenuated. In vessels treated with 8-BrcAMP (3 x 10(-4) M), 4-aminopyridine but not charybdotoxin inhibited relaxation induced by PTHrP 1-34 in both arteries and veins. Radioimmunoassay showed that, in the presence of a general phosphodiesterase inhibitor, PTHrP caused a concentration-dependent increase in intracellular cAMP content in arteries and veins, which was largely abolished by SQ-22536. Our results demonstrate that PTHrP is a potent vasodilator of pulmonary vessels, with a greater effect in veins than in arteries. Relaxation induced by the peptide contains both cAMP-dependent and -independent components. In both arteries and veins, voltage-dependent potassium channels mediate the response to PTHrP, at least in part, in a cAMP-independent fashion; and in veins, calcium-activated potassium channels may be stimulated by elevated cAMP levels.     

Diffusive oxygen shunting between vessels in the preglomerular renal vasculature: anatomic observations and computational modeling

To understand how geometric factors affect arterial-to-venous (AV) oxygen shunting, a mathematical model of diffusive oxygen transport in the renal cortex was developed. Preglomerular vascular geometry was investigated using light microscopy (providing vein shape, AV separation, and capillary density near arteries) and published micro-computed tomography (CT) data (providing vessel size and AV separation; Nordsletten DA, Blackett S, Bentley MD, Ritman EL, Smith NP. IUPS Physiome Project. http://www.physiome.org.nz/publications/nordsletten_blackett_ritman_bentley_smith_2005/folder_contents). A "U-shaped" relationship was observed between the arterial radius and the distance between the arterial and venous lumens. Veins were found to partially wrap around the artery more consistently for larger rather than smaller arteries. Intrarenal arteries were surrounded by an area of fibrous tissue, lacking capillaries, the thickness of which increased from ～5 μm for the smallest arteries (<16-μm diameter) to ～20 μm for the largest arteries (>200-μm diameter). Capillary density was greater near smaller arteries than larger arteries. No capillaries were observed between wrapped AV vessel pairs. The computational model comprised a single AV pair in cross section. Geometric parameters critical in renal oxygen transport were altered according to variations observed by CT and light microscopy. Lumen separation and wrapping of the vein around the artery were found to be the critical geometric factors determining the amount of oxygen shunted between AV pairs. AV oxygen shunting increases both as lumen separation decreases and as the degree of wrapping increases. The model also predicts that capillaries not only deliver oxygen, but can also remove oxygen from the cortical parenchyma close to an AV pair. Thus the presence of oxygen sinks (capillaries or tubules) near arteries would reduce the effectiveness of AV oxygen shunting. Collectively, these data suggest that AV oxygen shunting would be favored in larger vessels common to the cortical and medullary circulations (i.e., arcuate and proximal interlobular arteries) rather than the smaller vessels specific to the cortical circulation (distal interlobular arteries and afferent arterioles).     

Significance of source and size in the mechanical response of human cerebral blood vessels

Cerebral blood vessels are frequently damaged in traumatic brain injury. Mechanical properties of fresh human cerebral vessels obtained through surgeries have been reported. Because surgical sources of human specimens are rare and produce a limited amount of material, we sought to compare the properties of more readily available cerebral arteries and veins obtained from cadavers to fresh vessel data. Additionally, because the previous study was limited to small vessels available in surgery, it was unknown how generally applicable the results were to larger cerebral arteries and veins. In the current study, large and small cerebral vessels from autopsy were stretched axially. Data from these and similar tests on fresh vessels were combined to determine the significance of source and size on mechanical properties. Structural comparisons of histological samples were additionally utilized to characterize differences. Results indicate that specimens from autopsy and surgery behave similarly except that vessels from autopsy tend to be less extensible. While tests on large vessels were limited, small arteries obtained from autopsy tended to be slightly stiffer than large arteries. In contrast, bridging veins from cadavers were typically stiffer and stretched less before structural failure than cortical veins from the same source. These effects are, however, secondary to differences identified between arteries and veins in the previous study.     

Artery and vein size is balanced by Notch and ephrin B2/EphB4 during angiogenesis

A mutual coordination of size between developing arteries and veins is essential for establishing proper connections between these vessels and, ultimately, a functional vasculature; however, the cellular and molecular regulation of this parity is not understood. Here, we demonstrate that the size of the developing dorsal aorta and cardinal vein is reciprocally balanced. Mouse embryos carrying gain-of-function Notch alleles show enlarged aortae and underdeveloped cardinal veins, whereas those with loss-of-function mutations show small aortae and large cardinal veins. Notch does not affect the overall number of endothelial cells but balances the proportion of arterial to venous endothelial cells, thereby modulating the relative sizes of both vessel types. Loss of ephrin B2 or its receptor EphB4 also leads to enlarged aortae and underdeveloped cardinal veins; however, endothelial cells with venous identity are mislocalized in the aorta, suggesting that ephrin B2/EphB4 signaling functions distinctly from Notch by sorting arterial and venous endothelial cells into their respective vessels. Our findings provide mechanistic insight into the processes underlying artery and vein size equilibration during angiogenesis.     

Differentiation of the smooth muscle cell phenotypes during embryonic development of coronary vessels in the rat

Smooth muscle cell (SMC) maturation during embryonic development of coronary arteries and veins was studied in rats using different markers of the contractile phenotypes. The spatio-temporal pattern of distribution of these markers compared with the developing tunica media was examined. Alpha-smooth muscle actin (alpha-SMA) was the first marker of the SMC in the tunica media of coronary arteries found in ED16 hearts, followed by smooth muscle myosin heavy chain isoform which occurred on ED17. Subsequently 1E12 antigen was expressed in coronary artery wall in ED18 hearts, and finally smoothelin. The markers occur within the proximal part of the coronary arteries and deploy toward the apex. They are also found within the great vessels. None of the markers except for the alpha-SMA were found in coronary veins during embryonic life. We conclude that the SMC population of the developing tunica media of coronary vessels differentiates by the acquisition of particular markers and this process lasts till the end of the prenatal and early postnatal life.     

Molecular distinction between arteries and veins

The vertebrate vascular system is essential for the delivery and exchange of gases, hormones, metabolic wastes and immunity factors. These essential functions are carried out in large part by two types of anatomically distinct blood vessels, namely arteries and veins. Previously, circulatory dynamics were thought to play a major role in establishing this dichotomy, but recently it has become clear that arterial and venous endothelial cells are molecularly distinct even before the output of the first embryonic heartbeat, thus revealing the existence of genetic programs coordinating arterial-venous differentiation. Here we review some of the molecular mechanisms involved in this process.The first two authors contributed equally to this work     

Characteristics of myogenic reactivity in isolated rat mesenteric veins

Mechanisms of mechanically induced venous tone and its interaction with the endothelium and key vasoactive neurohormones are not well established. We investigated the contribution of the endothelium, l-type voltage-operated calcium channels (L-VOCCs), and PKC and Rho kinase to myogenic reactivity in mesenteric vessels exposed to increasing transmural pressure. The interaction of myogenic reactivity with norepinephrine (NE) and endothelin-1 (ET-1) was also investigated. Pressure myography was used to study isolated, cannulated, third-order rat mesenteric small veins and arteries. NE and ET-1 concentration response curves were constructed at low, intermediate, and high transmural pressures. Myogenic reactivity was not altered by nitric oxide synthase inhibition with N(ω)-nitro-L-arginine (L-NNA; 100 μM) or endothelium removal in both vessels. L-VOCCs blockade (nifedipine, 1 μM) completely abolished arterial tone, while only partially reducing venous tone. PKC (chelerythrine, 2.5 μM) and Rho kinase (Y27632, 3 μM) inhibitors largely abolished venous and arterial myogenic reactivity. There was no significant difference in the sensitivity of NE or ET-1-induced contractions within vessels. However, veins were more sensitive to NE and ET-1 when compared with corresponding arteries at low, intermediate, and high transmural pressures, respectively. These results suggest that 1) myogenic factors are important contributors to net venous tone in mesenteric veins; 2) PKC and Rho activation are important in myogenic reactivity in both vessels, while l-VOCCs play a limited role in the veins vs. the arteries, and the endothelium does not appear to modulate myogenic reactivity in either vessel type; and 3) mesenteric veins maintain an enhanced sensitivity to NE and ET-1 compared with the arteries when studied under conditions of changing transmural distending pressure.     

Comparison of the numbers of plasmalemmal vesicles in arterial and venous endothelia of rats

The numbers of plasmalemmal vesicles in endothelial cells of rat blood vessels were determined on electron microscopic sections. In all vessels examined which included aorta and carotid and femoral arteries, vena cava and femoral vein, and lung and brain capillaries, the numbers were of the same order of magnitude. For arteries the numbers were about double those for the corresponding veins. About one-third of all vesicles could be stained with ruthenium red after its infusion into the vessels. The results make it improbable that differences in numbers of 'transport' vesicles in different types of blood vessel contribute significantly to the selective accumulation of atherogenic plasma proteins in arteries.